U.S. patent application number 12/455742 was filed with the patent office on 2010-04-22 for integrin modulators and methods for their use.
This patent application is currently assigned to Billings Pharmaceuticals, Inc.. Invention is credited to John B. Davidson, Ali S. Turgiev.
Application Number | 20100099917 12/455742 |
Document ID | / |
Family ID | 34964172 |
Filed Date | 2010-04-22 |
United States Patent
Application |
20100099917 |
Kind Code |
A1 |
Davidson; John B. ; et
al. |
April 22, 2010 |
Integrin modulators and methods for their use
Abstract
1,5-Dithiaocta-2,7-diene-5-oxide-1-yl(DODOyl) compounds and
derivatives thereof, referred to collectively as DODOyl-derived
compounds (DDCs), and chiral enantiomers and derivatives thereof,
are described, which are integrin modulators that modulate
integrin-mediated functions and/or processes. Pharmaceutical
compositions containing integrin modulators and chiral enantiomers
thereof, and methods for using integrin modulators and chiral
enantiomers thereof are further described.
Inventors: |
Davidson; John B.; (Chicago,
IL) ; Turgiev; Ali S.; (Moscow, RU) |
Correspondence
Address: |
BRINKS HOFER GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Assignee: |
Billings Pharmaceuticals,
Inc.
|
Family ID: |
34964172 |
Appl. No.: |
12/455742 |
Filed: |
June 5, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10592212 |
Sep 8, 2006 |
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PCT/US2005/007638 |
Mar 10, 2005 |
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12455742 |
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60552239 |
Mar 10, 2004 |
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Current U.S.
Class: |
568/36 |
Current CPC
Class: |
C07C 323/65 20130101;
A61P 35/00 20180101; A61P 31/18 20180101 |
Class at
Publication: |
568/36 |
International
Class: |
C07C 317/08 20060101
C07C317/08 |
Claims
1-108. (canceled)
109. An integrin-modulator comprising: a compound that forms at
least one moiety in vivo, wherein the moiety has a structure:
##STR00050## wherein: X.sup.1 is selected from the group consisting
of hydrogen, an alkyl, an alkenyl, an alkynyl, an aryl, an aryl
substituted with one or more --NO.sub.2 groups, an aryl substituted
with one or more lower alkyls, a group of formula RO--, a group of
formula RC(O)--, a group of formula RC(O)O--, a group of formula
ROC(O)--, a group of formula (R).sub.2N--, a group of formula
RC(O)N--, a group of formula R(NH)C(O)--, a group of formula
RN.dbd.N--, a group of formula RS--, a group of formula
RSO.sub.2--, a group of formula RS(O)--, RSC(O)--, a group of
formula RSO2O--, a group of formula RS(O)O--, a halogen atom, a
nitroso group, a furanose unit, a pyranose unit, a combination of
furanose units, a combination of pyranose units, a combination of
furanose and pyranose units, and combinations thereof; R is
selected from the group consisting of a hydrogen, a lower alkyl, a
lower alkenyl, an aryl, an aryl substituted with one or more lower
alkyls, S-cysteinyl, peptidyl, an alkylphosphoglyceryl, an
alkenylphosphoglyceryl, or an acylphosphoglyceryl; m is an integer
from 0 to 30; and n is an integer from 0 to 30; providing that the
compound is not ajoene or ajocysteine.
110. The invention of claim 109, wherein X.sup.1 is alkenyl.
111. The invention of claim 109, wherein X.sup.1 is
CH.sub.2.dbd.CH--CH.sub.2--.
112. The invention of claim 109, wherein the radical is a (E,R)
stereoisomer.
113. The invention of claim 109, wherein the radical is a (E,S)
stereoisomer.
114. The invention of claim 109, wherein the radical is a (Z,R)
stereoisomer.
115. The invention of claim 109, wherein the radical is a (Z,S)
stereoisomer.
116. The invention of claim 109, wherein the compound comprises at
least two stereoisomers having a configuration selected from the
group consisting of (E,R), (E,S), (Z,R), (Z,S), and combinations
thereof.
117-173. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to agents for modulating one
or more integrin-mediated functions, and to methods for their
use.
BACKGROUND
[0002] Integrins are heterodimeric transmembrane glycoproteins
which, inter alia, act as cell receptors for various entities,
herein termed collectively "integrin ligands," including, for
example, surface molecules of other cells and extracellular matrix
(ECM) proteins. Both soluble and immobilized integrin ligands are
known to be ordinarily bound by integrins. Integrins are found on
most types of cells. Various cellular events in which integrins may
participate or which are associated with integrins or integrin
activity are referred to collectively herein as "integrin-mediated"
events. For a general review of integrins, see: Guidebook to the
Extracellular Matrix and Adhesion Proteins (Kreis et al., Eds.,
1993), and The Adhesion Molecule Facts Book (Pigot et al., Academic
Press, 1993).
[0003] One such integrin-mediated cellular function is signaling.
For instance, certain integrins are known to participate in the
transfer of information from the inside to the outside of the cell
(inside-out signaling) or from the outside to the inside of the
cell (outside-in signaling), although other types of signaling may
also occur, as may combinations thereof. An example involving
inside-out signaling is the process whereby an integrin acquires or
expresses affinity for ligands in response to intracellular events
(integrin upregulation). Binding of integrin ligands to certain
integrins (e.g., in the case of integrin-mediated cell adhesion)
may initiate signal transduction events, in a manner similar to
that described for other cell surface receptors. Signals thus
elicited are termed outside-in signals and are involved in the
regulation of various cell responses, which may include gene
expression, cell differentiation, and cell proliferation.
[0004] Signaling may result in the clustering of cellular molecules
in localized areas of cellular membrane, for example, in the
association of integrins with each other (and other molecules) by
interactions. The formation of such clusters may influence various
integrin-mediated functions in multiple ways, including, for
example, by additional or secondary signaling events or
interactions, and by altered ligand affinity.
[0005] The integrin-mediated function of adhesion is, or various
integrin-mediated events associated with adhesion are, important
for a variety of physiological and pathological responses. The
extent of adhesion is functionally related to integrin-mediated
signaling. For example, in association with initial
integrin-dependent adhesion to a substratum, certain cells change
their shape and start spreading on the surface of the stratum,
employing integrins in the process of establishing new contacts
with the underlying proteins (e.g., extracellular matrix [ECM]
components). In motile cells, the whole array of integrin-mediated
events involving adhesion--initial contact, cell shape change, cell
spreading, and cell locomotion--is sometimes termed "the adhesion
cascade" (S. R. Sharar et al., "The Adhesion Cascade and
Anti-Adhesion Therapy: An Overview," Springer Semin. Immunopathol.
1995, 16, 359). Adhesion cascades are viewed as integral to one or
more familiar cell motility patterns, including angiogenesis,
lymphocyte homing, tumor cell metastasis, and cell migration
processes associated with wound healing, although similar cascade
mechanisms are also viewed as operative even in the absence of cell
locomotion (e.g., in platelet adhesion and aggregation).
Extravasation (diapedesis) of neutrophils is described below in
greater detail, as a paradigmatic integrin-mediated adhesion
cascade (E. Hub et al., "Mechanism of Chemokine-Induced Leukocyte
Adhesion and Emigration," Chemoattractant Ligands and Their
Receptors (R. Horuk, Ed., CRC Press, Boca Raton, 1996, 301).
[0006] The onset of extravasation is heralded by the appearance in
the circulation of chemotactic factors, or chemoattractants (i.e.,
specific substances that initiate cell migration along their
concentration gradients). Chemoattractants (e.g., chemokines,
bacterial peptides, and products of complement activation) activate
neutrophils to upregulate their integrin receptors (neutrophil
integrins include, for example, LFA-1 [CD11a/CD18], CR3 [also known
as Mac-1, CD11b/CD18], and gp154,95 [CD11c/CD18]). Neutrophils thus
activated adhere to endotheliocytes, change shape, and spread on
the endothelial surface. Thereafter, the stimulated motile
apparatus of the neutrophils gives rise to migration, and the
neutrophils start moving, first across the endothelial layer and
further, through the perivascular ECM, towards the source of the
chemotactic stimulus (e.g., pathogenic bacteria invading a certain
bodily tissue). During the whole process, from the initial firm
contact with the endothelium to the cessation of locomotion at the
destination site, various integrins participate in the attachment
of the neutrophil to the substrata it encounters, enabling its
recruitment to the locus of infection. Parallel processes utilizing
integrins are involved when cancer cells migrate to initiate
metathetic foci in the body.
[0007] Another integrin-mediated function is cell-cell fusion.
Under physiological conditions, fusion is a developmentally
regulated stage in the differentiation of certain multinucleate
cells (e.g., osteoclasts, myocytes, and syncytiotrophoblasts), and
fusion is also a prerequisite to fertilization (in the case of
sperm-egg fusion). Fusion is effected by specialized cellular
systems involving integrins (e.g., refs. cited in: A.-P. J.
Huovila, et al., "ADAMs and Cell Fusion," Current Opin. Cell. Biol.
1996, 8, 692 and S. Ohgimoto et al., "Molecular Characterization of
Fusion Regulatory Protein-1 [FRP-1] that Induces Multinucleate
Giant Cell Formation of Monocytes and HIV gp160-Mediated Cell
Fusion: FRP-1 and 4F2/CD98 Are Identical Molecules," J. Immunol.
1995, 155, 3585).
[0008] The ability to undergo recirculation from intracellular
compartments to the cell surface and vice versa is a common
property of diverse cellular receptors, including integrins and
integrin components (e.g., see: P. Handagama et al., "Kistrin, an
Integrin Antagonist, Blocks Endocytosis of Fibrinogen into
Guinea-Pig Megakaryocyte and Platelet alpha-Granules," J. Clin.
Invest. 1993, 91, 193). This capability facilitates the mediation
of other cellular functions by transporting into the cell
extracellular material (e.g., soluble proteins, particulate matter,
and other cells). Integrin-mediated internalization is used by
certain microorganisms to invade their targets. For example, CR3
mediates entry of iC3b-opsonized HIV-1 and HIV-2 into CD4-negative
lymphocytic and monocytic cells (V. Boyer et al., "Complement
Mediates Human Immunodeficiency Virus Type 1 Infection of a Human T
cell Line in a CD4- and Antibody-Independent Fashion," J. Exp. Med.
1991, 173, 1151).
[0009] The above-delineated integrin-mediated functions are
illustrative only, as other characterizations of integrin-mediated
functions can also be made. Moreover, the integrin-mediated
functions as delineated herein are overlapping and interrelated. In
the case of neutrophil extravasation, for example, the initial
chemotactic signal activating the cells is commonly functionally
associated with integrin upregulation (inside-out signaling) and
adhesion to the endothelial surface. This adhesion event, in turn,
is associated with an outside-in signal, enabling the neutrophil to
change its shape, which is a prerequisite to the spreading and
migration of the cell. Likewise, when the neutrophil that has
arrived to the source of chemoattractants establishes an adhesive
interaction with the bacteria by means of integrins, an outside-in
signal is transduced, which is associated with the initiation of
internalization of the integrins involved, together with the
bacteria attached thereto (phagocytosis).
[0010] Furthermore, regarding outside-in integrin-mediated
signaling, certain cellular processes are co-mediated by several
signaling systems acting in concert. In the case of neutrophils
extravasating to the tissues to phagocytose bacteria, the
neutrophils receive signals by means of the receptors of the
chemoattractant (along the concentration gradient of which the
movement occurs) and by means of distinct integrins (including
those that attach it to the substratum and, subsequently, those
recognizing the bacteria). This interplay of signals mediates the
antibacterial machinery of the neutrophils with the consequence
that only upon contact with the bacteria, which is mediated by
means of a particular type of integrin, are the constituents of the
intracellular granules released and reactive oxygen species formed.
As a result, the formation and release of microbicidal substances
take place preferentially at sites of contact with bacteria,
enabling effective killing of the bacteria and preventing the
destruction of host tissue (S. D. Wright, "Receptors for Complement
and the Biology of Phagocytosis" in Inflammation: Basic Principles
and Clinical Correlates, 2.sup.nd Ed. (J. I. Gallin et al., Eds.,
Raven Press, New York, 1992, Chapter 25, 477).
[0011] A broad range of cellular activities can be regulated by
modulating certain integrin-mediated functions with appropriate
agents. One such modulating agent is ajoene
(4,5,9-trithiadodeca-1,6,11-triene-9-oxide):
##STR00001##
[0012] Ajoene, and a precursor thereof, can be formed via reactions
involving products derived from garlic (Allium sativum). As an
example, when garlic is crushed, cellular alliin may come into
contact with alliinase in the cell wall to form allicin. In the
presence of an appropriate polar solvent, allicin may then form
ajoene. Ajoene has been previously shown to inhibit platelet
aggregation by allosterically inactivating the platelet integrin,
GP IIb/IIIa (R. Apitz-Castro et al., Biophys. Res. Commun. 1986,
141, 145). It has been demonstrated that stereoisomers at the
internal double bond of ajoene (i.e., E- and
Z-4,5,9-trithiadodeca-1,6,11-triene-9-oxides) exhibit no
significant differences in their effects on platelets (E. Block et
al., J. Am. Chem. Soc. 1986, 108, 7045). For this reason, most of
the subsequent studies of the integrin modulation by ajoene were
carried out on mixtures (typically designated as racemic) of the E-
and Z-isomers. It was shown, for example, that ajoene is a potent
inhibitor of a wide variety of adhesion-dependent processes,
including neutrophil aggregation, HIV transmission (A. V.
Tatarintsev et al., AIDS 1992, 6, 1215), and tumor metastasis.
United States patents issued to Tatarintsev et al. describe the use
of ajoene for treatment of inflammation (U.S. Pat. No. 5,948,821),
arthritis (U.S. Pat. No. 5,856,363), and tumors (U.S. Pat. No.
5,932,621), as well as for contraception (U.S. Pat. No. 5,863,954)
and inhibition of immune responses (U.S. Pat. No. 5,863,955). All
of these diseases and conditions are believed to involve
integrin-mediated processes. Ajoene has also been used to treat
additional diseases and conditions which are believed to involve
integrin-mediated processes, as described in PCT application WO
97/25031.
[0013] The presence in ajoene of the 6-double bond generates,
depending on its geometry, two stereoisomers, E- and Z-ajoenes. The
presence of the chiral 9-sulfoxide group generates the possibility
for two optical isomers (enantiomers) of each stereoisomer. Thus,
the above structural features of ajoene create the possibility for
four enantiomers: (6E,9R), (6E,9S), (6Z,9R), and
(6Z,9S)-4,5,9-trithiadodeca-1,6,11-triene-9-oxides. However, in the
case of allicin (which also contains a sulfoxide functionality),
the existence of optical activity has been questioned, so that the
existence of enantiomers, or at least stable enantiomers, would
have been considered unlikely (L. D. Lawson et al., Eds., "Garlic:
The Science and Therapeutic Application of Allium Sativum L. and
Related Species," 1997, page 56).
SUMMARY
[0014] The scope of the present invention is defined solely by the
appended claims, and is not affected to any degree by the
statements within this summary.
[0015] By way of introduction, a first compound embodying features
of the present invention has a structure:
##STR00002##
[0016] wherein:
[0017] X.sup.1 and X.sup.2 are the same or different and are each
independently selected from the group consisting of hydrogen, an
alkyl, an alkenyl, an alkynyl, an aryl, an aryl substituted with
one or more --NO.sub.2 groups, an aryl substituted with one or more
lower alkyls, a group of formula RO--, a group of formula RC(O)--,
a group of formula RC(O)O--, a group of formula ROC(O)--, a group
of formula (R).sub.2N--, a group of formula RC(O)N--, a group of
formula R(NH)C(O)--, a group of formula RN.dbd.N--, a group of
formula RSO.sub.2--, a group of formula RS(O)--, RSC(O)--, a group
of formula RSO2O--, a group of formula RS(O)O--, a halogen atom, a
nitroso group, a furanose unit, a pyranose unit, a combination of
furanose units, a combination of pyranose units, a combination of
furanose and pyranose units, and combinations thereof;
[0018] R is independently in each occurrence selected from the
group consisting of a hydrogen, a lower alkyl, a lower alkenyl, an
aryl, an aryl substituted with one or more lower alkyls,
S-cysteinyl, peptidyl, an alkylphosphoglyceryl, an
alkenylphosphoglyceryl, or an acylphosphoglyceryl;
[0019] m is an integer from 0 to 30; and n is an integer from 0 to
30;
[0020] providing that when X.sup.1 is CH.sub.2.dbd.CH--CH.sub.2--,
m is 1, and n is 0, X.sup.2 is not CH.sub.2.dbd.CH--CH.sub.2--S--
or (NH.sub.2)--CH(CO.sub.2H)--CH.sub.2--S--.
[0021] A second compound embodying features of the present
invention has a structure:
##STR00003##
[0022] wherein:
[0023] X.sup.1 is selected from the group consisting of hydrogen,
an alkyl, an alkenyl, an alkynyl, an aryl, an aryl substituted with
one or more --NO.sub.2 groups, an aryl substituted with one or more
lower alkyls, a group of formula RO--, a group of formula RC(O)--,
a group of formula RC(O)O--, a group of formula ROC(O)--, a group
of formula (R).sub.2N--, a group of formula RC(O)N--, a group of
formula R(NH)C(O)--, a group of formula RN.dbd.N--, a group of
formula RS--, a group of formula RSO.sub.2--, a group of formula
RS(O)--, RSC(O)--, a group of formula RSO2O--, a group of formula
RS(O)O--, a halogen atom, a nitroso group, a furanose unit, a
pyranose unit, a combination of furanose units, a combination of
pyranose units, a combination of furanose and pyranose units, and
combinations thereof;
[0024] X.sup.2 is a polymeric species comprising a plurality of
binding sites;
[0025] R is independently in each occurrence selected from the
group consisting of a hydrogen, a lower alkyl, a lower alkenyl, an
aryl, an aryl substituted with one or more lower alkyls,
S-cysteinyl, peptidyl, an alkylphosphoglyceryt, an
alkenylphosphoglyceryl, or an acylphosphoglyceryl; and
m is an integer from 0 to 30; n is an integer from 0 to 30; and w
is an integer from 2 to 1000.
[0026] A third compound embodying features of the present invention
has a structure:
##STR00004##
[0027] wherein X.sup.1 and X.sup.2 are the same or different and
comprise at least one functional group configured for participation
in a polymerization reaction;
[0028] wherein m is an integer from 0 to 30;
[0029] wherein n is an integer from 0 to 30; and
[0030] wherein w is an integer from 2 to 1000.
[0031] A fourth compound embodying features of the present
invention has a structure:
##STR00005##
[0032] wherein X.sup.1 comprises at least one functional group
configured for participation in a polymerization reaction;
[0033] wherein m is an integer from 0 to 30;
[0034] wherein n is an integer from 0 to 30; and
[0035] wherein w is an integer from 2 to 1000.
[0036] A fifth compound embodying features of the present invention
has a structure:
##STR00006##
[0037] wherein X.sup.1 and X.sup.2 are the same or different and
are each independently selected from the group consisting of
hydrogen, an alkyl, an alkenyl, an alkynyl, an aryl, an aryl
substituted with one or more --NO.sub.2 groups, an aryl substituted
with one or more lower alkyls, a group of formula RO--, a group of
formula RC(O)--, a group of formula RC(O)O--, a group of formula
ROC(O)--, a group of formula (R).sub.2N--, a group of formula
RC(O)N--, a group of formula R(NH)C(O)--, a group of formula
RN.dbd.N--, a group of formula RSO.sub.2--, a group of formula
RS(O)--, RSC(O)--, a group of formula RSO.sub.2O--, a group of
formula RS(O)O--, a halogen atom, a nitroso group, a furanose unit,
a pyranose unit, a combination of furanose units, a combination of
pyranose units, a combination of furanose and pyranose units, and
combinations thereof;
[0038] R is independently in each occurrence selected from the
group consisting of a hydrogen, a lower alkyl, a lower alkenyl, an
aryl, an aryl substituted with one or more lower alkyls,
S-cysteinyl, peptidyl, an alkylphosphoglyceryl, an
alkenylphosphoglyceryl, or an acylphosphoglyceryl;
[0039] m is an integer from 0 to 30;
[0040] n is an integer from 0 to 30;
[0041] o is an integer from 0 to 30; and
[0042] p is an integer from 0 to 30.
[0043] A sixth compound embodying features of the present invention
has a structure:
##STR00007##
[0044] wherein X.sup.1 and X.sup.2 are the same or different and
are each independently selected from the group consisting of
hydrogen, an alkyl, an alkenyl, an alkynyl, an aryl, an aryl
substituted with one or more --NO.sub.2 groups, an aryl substituted
with one or more lower alkyls, a group of formula RO--, a group of
formula RC(O)--, a group of formula RC(O)O--, a group of formula
ROC(O)--, a group of formula (R).sub.2N--, a group of formula
RC(O)N--, a group of formula R(NH)C(O)--, a group of formula
RN.dbd.N--, a group of formula RSO.sub.2--, a group of formula
RS(O)--, RSC(O)--, a group of formula RSO.sub.2O--, a group of
formula RS(O)O--, a halogen atom, a nitroso group, a furanose unit,
a pyranose unit, a combination of furanose units, a combination of
pyranose units, a combination of furanose and pyranose units, and
combinations thereof;
[0045] R is independently in each occurrence selected from the
group consisting of a hydrogen, a lower alkyl, a lower alkenyl, an
aryl, an aryl substituted with one or more lower alkyls,
S-cysteinyl, peptidyl, an alkylphosphoglyceryl, an
alkenylphosphoglyceryl, or an acylphosphoglyceryl;
[0046] X.sup.3 is selected from the group consisting of an alkylene
group and a metal atom;
[0047] m is an integer from 0 to 30;
[0048] n is an integer from 0 to 30;
[0049] o is an integer from 0 to 30; and
[0050] p is an integer from 0 to 30.
[0051] A pharmaceutical composition embodying features of the
present invention includes a pharmaceutically-acceptable carrier
and at least one of the compounds described above.
[0052] A method of modulating an integrin-mediated function of one
or more cells that embodies features of the present invention
includes contacting at least one of the cells with at least one of
the compounds described above in an amount effective to modulate
the integrin-mediated function.
[0053] A method of treating or preventing a disorder, disease or
condition involving an integrin-mediated function that embodies
features of the present invention includes administering to an
animal in need thereof an effective amount of at least one of the
compounds described above.
[0054] A method of treating or preventing at least one of a
thrombotic disorder, disease or condition arising therefrom that
embodies features of the present invention includes administering
to an animal in need thereof an effective amount of at least one of
the compounds described above.
[0055] A method of treating or preventing inflammation or an
inflammatory disease that embodies features of the present
invention includes administering to an animal in need thereof an
effective amount of at least one of the compounds described
above.
[0056] A method of treating, preventing, or inhibiting transmission
of a viral infection that embodies features of the present
invention includes administering to an animal in need thereof an
effective amount of at least one of the compounds described
above.
[0057] A method of treating psoriasis that embodies features of the
present invention includes administering to an animal in need
thereof an effective amount of at least one of the compounds
described above.
[0058] A method of treating atherosclerosis that embodies features
of the present invention includes administering to an animal in
need thereof an effective amount of at least one of the compounds
described above.
[0059] A method of treating cancer, preventing metastasis of
tumors, or inhibiting integrin-mediated carcinogenesis that
embodies features of the present invention includes administering
to an animal in need thereof an effective amount of at least one of
the compounds described above.
[0060] An integrin-modulator embodying features of the present
invention includes a compound that forms at least one moiety in
vivo, wherein the moiety has a structure:
##STR00008##
[0061] wherein:
[0062] X.sup.1 is selected from the group consisting of hydrogen,
an alkyl, an alkenyl, an alkynyl, an aryl, an aryl substituted with
one or more --NO.sub.2 groups, an aryl substituted with one or more
lower alkyls, a group of formula RO--, a group of formula RC(O)--,
a group of formula RC(O)O--, a group of formula ROC(O)--, a group
of formula (R).sub.2N--, a group of formula RC(O)N--, a group of
formula R(NH)C(O)--, a group of formula RN.dbd.N--, a group of
formula RS--, a group of formula RSO.sub.2--, a group of formula
RS(O)--, RSC(O)--, a group of formula RSO2O--, a group of formula
RS(O)O--, a halogen atom, a nitroso group, a furanose unit, a
pyranose unit, a combination of furanose units, a combination of
pyranose units, a combination of furanose and pyranose units, and
combinations thereof;
[0063] R is selected from the group consisting of a hydrogen, a
lower alkyl, a lower alkenyl, an aryl, an aryl substituted with one
or more lower alkyls, S-cysteinyl, peptidyl, an
alkylphosphoglyceryl, an alkenylphosphoglyceryl, or an
acylphosphoglyceryl;
[0064] m is an integer from 0 to 30; and
[0065] n is an integer from 0 to 30;
[0066] providing that the compound is not ajoene or
ajocysteine.
[0067] A liposome preparation embodying features of the present
invention includes a compound having a formula:
##STR00009##
[0068] wherein X.sup.1 and X.sup.2 are the same or different and
include at least one functional group for binding to at least one
of a phospholipid, a glycolipid, and a sterol;
[0069] wherein m is an integer from 0 to 30;
[0070] wherein n is an integer from 0 to 30;
[0071] wherein w is an integer from 2 to 1000; and
[0072] wherein a lipid bound to the compound is adapted for
incorporation into liposomes or micelles.
BRIEF DESCRIPTION OF THE DRAWING
[0073] FIG. 1 shows a graph of HIV-induced syncytium formation as a
percentage of untreated control versus concentration (micromoles
per liter) of the unseparated 3:1 mixture of racemic (Z)-(.+-.)-
and racemic (E)-(.+-.)-ajoenes (.diamond-solid.), (Z)-(+)-ajoene
(+), racemic (E)-(.+-.)-ajoene (o), and (Z)-(-)-ajoene (x) (curves
1 through 4, left to right, respectively).
DETAILED DESCRIPTION
[0074] It has been discovered that the activity of ajoene as a
chiral integrin modulator (CIM) may be traced to its ability to
form in vivo one or a plurality of active moieties having the
formula:
##STR00010##
which include the (2E)-(+)-, (2E)-(-)-, (2Z)-(+)-, and (2Z)-(-)
isomers. The active moiety shown above includes a
1,5-dithiaocta-2,7-diene-5-oxide-1-yl fragment (hereinafter
DODOyl), which is incorporated into integrin modulators in
accordance with the present invention, as further described
below.
[0075] In accordance with this discovery, integrin modulators have
been discovered that have a formula:
##STR00011##
[0076] wherein:
[0077] X.sup.1 is selected from the group consisting of hydrogen,
an alkyl, an alkenyl, an alkynyl, an aryl, an aryl substituted with
one or more --NO.sub.2 groups, an aryl substituted with one or more
lower alkyls, a group of formula RO--, a group of formula RC(O)--,
a group of formula RC(O)O--, a group of formula ROC(O)--, a group
of formula (R).sub.2N--, a group of formula RC(O)N--, a group of
formula R(NH)C(O)--, a group of formula RN.dbd.N--, a group of
formula RS--, a group of formula RSO.sub.2--, a group of formula
RS(O)--, a group of formula RSC(O)--, a group of formula
RSO.sub.2O--, a group of formula RS(O)O--, a halogen atom, a
nitroso group, a furanose unit, a pyranose unit, a combination of
furanose units, a combination of pyranose units, a combination of
furanose and pyranose units, and combinations thereof;
[0078] R is selected from the group consisting of a hydrogen, a
lower alkyl, a lower alkenyl, an aryl, an aryl substituted with one
or more lower alkyls, S-cysteinyl, peptidyl, an
alkylphosphoglyceryl, an alkenyiphosphoglyceryl, or an
acylphosphoglyceryl;
[0079] m is an integer from 0 to 30; and
[0080] n is an integer from 0 to 30.
[0081] In further accordance with this discovery, integrin
modulators have been discovered that have a formula:
##STR00012##
[0082] wherein X.sup.1, R, m, and n are as defined above for the
corresponding thia radical.
[0083] In further accordance with this discovery, integrin
modulators have been discovered that include compounds capable of
forming an active moiety analogous to the DODOyl fragment shown
above, as well as products formed therefrom (e.g., by
bond-formation and/or rearrangement). Integrin modulators embodying
features of the present invention include DODOyl compounds and
derivatives thereof, hereinafter referred to collectively as
DODOyl-derived compounds (DDCs), which have a general formula:
##STR00013##
wherein:
[0084] X.sup.1 and X.sup.2 are the same or different and are each
independently selected from the group defined above for the X.sup.1
portion of the corresponding thia radical;
[0085] R is independently in each occurrence selected from the
group defined above for the R group of the corresponding thia
radical; and
[0086] m and n are as defined above for the corresponding thia
radical;
[0087] providing that when X.sup.1 is CH.sub.2.dbd.CH--CH.sub.2--,
m is 1, and n is 0, X.sup.2 is not CH.sub.2.dbd.CH--CH.sub.2--S--
or (NH.sub.2)--CH(CO.sub.2H)--CH.sub.2--S--.
[0088] In the above-described formula, m can also be an integer
from 0 to about 3, and n can also be an integer from 0 to about 3.
In a first series of presently preferred DDCs embodying features of
the present invention, m=1 and n=0. In this first series, it is
presently preferred that X.sup.1 is alkenyl. Moreover, it is
presently preferred that at least one double bond in the alkenyl
X.sup.1 fragment be allylic to the sulfoxide group in order to
resemble the structure of the terminal double bond allylic to the
sulfoxide group in ajoene. One presently preferred alkenyl group
corresponds to X.sup.1 is allyl. In a second series of presently
preferred DDCs embodying features of the present invention, m=0 and
n=1.
[0089] When X.sup.1 is allyl, m=1, and n=0, the formula above
corresponds to the prototypal DODOyl substituted compounds, which
constitute a presently preferred DDC subgroup in accordance with
the present invention. X.sup.2 in this subgroup may correspond to
any of the groups described above with the exception of
CH.sub.2.dbd.CH--CH.sub.2--S-- and
(NH.sub.2)--CH(CO.sub.2H)--CH.sub.2--S--. As described above, it is
presently preferred that the X.sup.1 portion of DDCs in accordance
with the present invention be structurally analogous to the allyl
group attached to the sulfoxide in ajoene. However, since the
X.sup.2 portion at the other end of the DDC may be cleaved to
generate a thia moiety, as described above, a wide array of
functionalities may be included therein.
[0090] Yet another distinct DDC subgroup embodying features of the
present invention includes compounds which share two common
characteristics: (a) the polymeric nature of X.sup.2 and (b)
multiplicity of the active chiral moieties attached to a single
X.sup.2. These compounds have a general formula:
##STR00014##
[0091] wherein X.sup.1, m, and n are as defined above for the
corresponding thia radical;
[0092] wherein X.sup.2 is a polysaccharide, polypeptide, or other
suitable substrate; and
[0093] w is an integer from 7 to 1000.
[0094] Polymeric DDCs embodying features of the present invention
may also have a formula:
##STR00015##
[0095] wherein X.sup.1 and X.sup.2 may be the same or different and
are preferentially selected from functionalities enabling
introduction of the chiral moiety into a phospholipid, glycolipid,
or sphingolipid; wherein m and n are as defined above for the
corresponding thia radical; and wherein w is an integer from 7 to
1000. Incorporation of such lipids into liposomes or micelles
results in a subgroup of DDCs that are both polymeric (i.e., each
liposome carries a desired amount of DDCs) and particulate (i.e.,
suspension of liposomes).
[0096] In addition, polymeric DDCs embodying features of the
present invention may also have a formula:
##STR00016##
[0097] wherein X.sup.1 and X.sup.2 are the same or different and
comprise at least one functional group configured for participation
in a polymerization reaction;
[0098] wherein m is an integer from 0 to 30;
[0099] wherein n is an integer from 0 to 30; and
[0100] wherein w is an integer from 2 to 1000.
[0101] Additional polymeric DDCs embodying features of the present
invention may have a formula:
##STR00017##
[0102] wherein X.sup.1 comprises at least one functional group
configured for participation in a polymerization reaction;
[0103] wherein m is an integer from 0 to 30;
[0104] wherein n is an integer from 0 to 30; and
[0105] wherein w is an integer from 2 to 1000.
[0106] It is understood that any DDC embodying features of the
present invention includes the E and the Z stereoisomers (generated
by the geometry of the central double bond), each having the R and
the S configurations (generated by the presence of the sulfoxide
functionality). The notations (E, R), (E, S), (Z, R), and (Z, S)
are used herein to denote geometric isomers and enantiomers of DDCs
embodying features of the present invention. Where applicable, as
described herein, the (-) sign below the sulfur atom denotes the
levorotatory or (-) enantiomer, and the (+) sign below the sulfur
atom describes the dextrorotatory or (+) enantiomer. As will be
understood by those of ordinary skill in the art, the optical
rotation designations + and - are determined on a case-by-case
basis for specific isomers using, for example, a polarimeter. It is
to be understood that DDCs in accordance with the present invention
may include one or more additional double bonds and/or additional
chiral centers in the X.sup.1 and/or X.sup.2 portions thereof.
Accordingly, when the E/Z designations are used herein to describe
DDCs in accordance with the present invention, it is to be
understood that these designations are used in reference to the
geometry of the central double bond drawn in the general formulae
(i.e., other double bonds in the molecule may have the same and/or
different geometry as the central double bond). Similarly, when the
R/S and/or (+)/(-)designations are used herein to describe DDCs in
accordance with the present invention, it is to be understood that
these designations are used in reference to the configuration of
the central sulfoxide functionality drawn in the general formulae
(i.e., additional chiral centers in the molecule may have the same
and/or different chiral designation as the central sulfoxide
group).
[0107] In view of the foregoing explanation, it is appreciated
that, in one illustrative aspect, the compounds described herein
can be mixtures containing all four enantiomers--that is, (E,R),
(E,S), (Z,R), and (Z,S). In another illustrative aspect, the
compounds described herein can be mixtures of two enantiomers
having one double bond geometry or the other--that is, (E,R) and
(E,S) or (Z,R) and (Z,S). In another illustrative aspect, the
compounds described herein can be mixtures of two stereoisomers
having the same sulfoxide chirality--that is, (E,R) and (Z,R) or
(E,S) and (Z,S). In another illustrative aspect, the compounds
described herein can be single enantiomers having one sulfoxide
chirality and one double bond geometry--that is, (E,R), (E,S),
(Z,R) or (Z,S). In another illustrative aspect, the compounds
described herein can be mixtures of two enantiomers not matching
each other in both the double bond geometry and the sulfoxide
chirality--that is, (E,R) and (Z,S) or (E,S) and (Z,R). In yet
another illustrative aspect, the compounds can be mixtures of any
three out of the four possible enantiomers.
[0108] The present invention also provides methods of using the
above-described DDCs.
[0109] In a first aspect, the present invention provides methods of
modulating an integrin-mediated function of one or more cells using
DDCs embodying features of the present invention.
[0110] In a second aspect, the present invention provides methods
for the treatment of a variety of disorders, diseases and
conditions. In particular, the present invention provides but is
not limited to: (a) methods of treating or preventing a disorder,
disease or condition in which one or more integrin-mediated
functions play a role; (b) methods of treating or preventing
thrombotic disorders and diseases/conditions arising therefrom
(e.g., embolism, ischemia, infarction, etc.); (c) methods of
treating or preventing inflammation and inflammatory diseases; (d)
methods of treating, preventing, or inhibiting the transmission of
viral infections; (e) methods of treating shock; (f) a method of
treating arthritis; (g) methods of contraception; (h) methods of
treating or suppressing adverse, undesirable or self-destructive
immune responses, including but not limited to acute and chronic
hypersensitivity reactions (such as anaphylaxis and allergy),
transplant rejection, and graft-versus-host disease (GVHD); (i)
methods of treating autoimmune diseases; (j) methods of inhibiting
undesirable integrin-mediated cell-cell fusion; (k) methods of
inhibiting the formation of lesions; (l) methods of treating
psoriasis; (m) methods of treating atherosclerosis; (n) methods of
treating diseases or conditions involving a plurality of
integrin-dependent etiopathogenetic mechanisms; (o) methods of
inhibiting the transfer of genetic material; (p) methods of
treating cancer, preventing metastasis of tumors, or inhibiting
certain (integrin-mediated) types of carcinogenesis; (q) methods of
treating or preventing diseases involving both autoimmune and
inflammatory conditions (e.g., diabetes, lupus, etc.); and the
like.
[0111] The present invention further provides pharmaceutical
compositions. The compositions include a DDC embodying features of
the present invention and a pharmaceutically-acceptable
carrier.
[0112] In addition, the present invention provides methods of
treating an organ or a tissue thereof by contacting the organ or
tissue with a DDC embodying features of the present invention. Such
treatment improves the condition of the organ or tissue for
subsequent use, as compared to organ or tissue which is not treated
with a DDC in accordance with the present invention. In particular,
an organ or tissue which is to be transplanted into a recipient may
be treated with a DDC prior to, in the course of, and/or after
harvesting, and/or prior to, in the course of, and/or after
transplantation, and the chances of the tissue being successfully
transplanted will be increased.
[0113] The present invention further provides kits for treating
tissue. The kits comprise one or more DDCs embodying features of
the present invention.
[0114] In addition, the present invention provides methods whereby
the integrin-modulating activity of DDCs may be assessed. One such
method involves VLA-4-mediated adhesion of enzyme-labeled PM1 cells
to VCAM-1-coated artificial substrata. Specifically, the cells are
exposed to isolated DDC enantiomers (or the vehicle thereof) and
allowed to adhere or not adhere to immobilized VCAM-1. Thereafter,
the adherent cells are lysed and, following addition of the
substrate of the enzyme and incubation, the activity of the enzyme
is measured spectrophotometrically. The value of this parameter,
characterizing the number of the adherent cells, is inversely
proportional to integrin-modulating activity of the compound.
Another such method is based on the inhibition of HIV-mediated
syncytium formation, a phenomenon known to depend on
integrin-mediated functions. Yet another method is based on the
inhibition of cell-to-cell HIV transmission, a process also
dependent on integrin-mediated functions.
[0115] CIMs based on enantiomers of ajoene and derivatives thereof
are described in U.S. patent application Ser. No. 10/332,545, filed
Jan. 9, 2003, International Application No. PCT/US01/21826, filed
Jul. 10, 2001, and U.S. Provisional Patent Application No.
60/217,651, filed Jul. 10, 2000. The entire contents of all of the
above-identified applications are incorporated herein by reference,
except that in the event of any inconsistent disclosure or
definition from the present application, the disclosure or
definition herein shall be deemed to prevail.
[0116] Throughout this description and the appended claims, the
following definitions are intended.
[0117] The phrase "integrin modulator" refers to an agent that
adjusts, varies, modifies, alters or affects an integrin-mediated
function. As used herein, the phrase "integrin modulator" includes
agents that act directly on integrins as well as agents that act
indirectly on integrins (e.g., by acting on one or more species
that themselves act on or in concert with integrins).
[0118] The term "racemic" is used to describe mixtures of optical
isomers of a chemical compound (e.g., dextrorotatory and
levorotatory), which may correspond to substantially equal amounts
thereof or to a preponderance of one or the other. As used herein,
the term "racemic" is also used to describe mixtures of E and Z
isomers of a double bond-containing compound (i.e., mixtures of E
and Z isomers), which may correspond to substantially equal amounts
thereof or to a preponderance of one or the other.
[0119] The term "tissue" is used in reference to both a
substantially complete organ (e.g., a heart) as well as to any
portion thereof (e.g., a ventricle). Thus, references herein to
methods of treating tissue are to be understood as including
methods for the treatment of organs as well.
[0120] The phrase "furanose unit" refers to a five-membered cyclic
form of pentose, hexose, or heptose, optionally modified with amino
groups, sulfhydryl groups, or residues of acetic, phosphoric,
and/or sulfuric acids.
[0121] The phrase "pyranose unit" refers to a six-membered cyclic
form of pentose, hexose, or heptose, optionally modified with amino
groups, sulfhydryl groups, or residues of acetic, phosphoric,
and/or sulfuric acids.
[0122] The related terms "polymer," "polymerization," and
"polymeric" refer to molecules, or to the preparation of molecules,
containing two or more monomeric units. Thus, as used herein, the
term "polymer" subsumes the term "oligomer."
[0123] The term "alkyl" refers to a straight-chain or
branched-chain hydrocarbon containing 1-30 carbon atoms, or a
cyclic hydrocarbon containing 3-20 carbon atoms. The alkyl may be
substituted or unsubstituted. The phrase "lower alkyl" refers a
straight-chain or branched-chain alkyl containing 1-4 carbon atoms.
Both terms include all possible isomers. Representative presently
preferred alkyls for use in accordance with the present invention
include but are not limited to methyl, ethyl, propyl, iso-propyl,
n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl,
neo-pentyl, n-hexyl, cyclohexyl, and the like.
[0124] The term "alkenyl" refers to a straight-chain or
branched-chain hydrocarbon containing 2-30 carbon atoms, or a
cyclic hydrocarbon containing 3-20 carbon atoms, and one or a
plurality of double bonds. The alkenyl may be substituted or
unsubstituted. The term includes all possible isomers.
Representative presently preferred alkenyls for use in accordance
with the present invention include but are not limited to vinyl,
propenyl, iso-propenyl, and the like.
[0125] The term "alkynyl" refers to a straight-chain or
branched-chain hydrocarbon containing 2-30 carbon atoms, or a
cyclic hydrocarbon containing 3-20 carbon atoms, and one or a
plurality of triple bonds. The alkynyl may be substituted or
unsubstituted. The term includes all possible isomers.
Representative presently preferred alkynyls for use in accordance
with the present invention include but are not limited to ethynyl,
1-propenyl, 2-propenyl, and the like.
[0126] The term "aryl" refers to a group containing at least one
aromatic ring, with phenyl being a presently preferred example. The
aryl may be substituted or unsubstituted. Electron-withdrawing
groups are presently preferred substituents. For example, the aryl
may be preferably substituted with one or more --NO.sub.2 groups,
in which case it is preferably m-nitrophenyl, p-nitrophenyl,
o-nitrophenyl, 3,5-dinitrophenyl, or 2,4-dinitrophenyl. The term
aryl also includes arylalkyl groups that may optionally be
substituted with one or more groups selected from alkyl and
--NO.sub.2. Representative presently preferred arylalkyls for use
in accordance with the present invention include but are not
limited to benzyl, o-tolyl, p-tolyl, m-tolyl, 3,5-xylyl, 2,6-xylyl,
and the like. The term also includes polycyclic aromatic groups.
Presently preferred polycyclic aromatic groups for use in
accordance with the present invention contain the cyclopentane
perhydrophenanthrene (steroid) backbone.
[0127] The term "halogen" refers to an atom in Group VIIB of the
periodic table (e.g., fluorine, chlorine, bromine, iodine,
astatine, etc.).
[0128] The term "peptidyl" refers to a straight-chain or a
branched-chain S-cysteinyl peptide function.
[0129] The term "alkylphosphoglyceryl" refers to
1-O-alkyl-2-O-sn-glyceroyl 3-O-phosphate,
2-O-alkyl-1-O-sn-glyceroyl 3-O-phosphate,
1-O-alkyl-2-N-iminoglyceryl 3-O-phosphate,
2-O-alkyl-1-N-iminoglyceryl 3-O-phosphate,
1-O-alkyl-2-S-thiaglyceryl 3-O-phosphate,
2-O-alkyl-1-S-thiaglyceryl 3-O-phosphate, and the like. The prefix
"alkyl" as applied to a phosphoglyceryl is to be understood as
defined above, with presently preferred alkyls including but not
limited to straight-chain or branched-chain hydrocarbons.
[0130] The term "alkenylphosphoglyceryl" refers to
1-O-alkenyl-2-O-sn-glyceroyl 3-O-phosphate,
2-O-alkenyl-1-O-sn-glyceroyl 3-O-phosphate,
1-alkenyl-1-hydroxy-2-N-iminoglyceryl 3-O-phosphate,
1-O-alkenyl-2-N-iminoglyceryl 3-O-phosphate,
2-O-alkenyl-1-N-iminoglyceryl 3-O-phosphate,
1-O-alkenyl-2-S-thiaglyceryl 3-O-phosphate,
2-O-alkenyl-1-S-thiaglyceryl 3-O-phosphate, and the like. The
prefix "alkenyl" as applied to a phosphoglyceryl is to be
understood as defined above, with presently preferred alkenyls
including but not limited to straight-chain or branched-Chain
hydrocarbons.
[0131] Unless noted otherwise, the variable "R" included in the
following definitions refers to hydrogen or to any substituted or
unsubstituted alkyl, alkenyl, alkynyl, or aryl moiety, wherein
alkyl, alkenyl, alkynyl, and aryl are as defined above.
[0132] The term "acylphosphoglyceryl" refers to
1-O-acyl-2-O-sn-glyceroyl 3-O-phosphate, 2-O-acyl-1-O-sn-glyceroyl
3-O-phosphate, 1-O-acyl-2-N-iminoglyceryl 3-O-phosphate,
2-O-acyl-1-N-iminoglyceryl 3-O-phosphate, 1-O-acyl-2-S-thiaglyceryl
3-O-phosphate, 2-O-acyl-1-S-thiaglyceryl 3-O-phosphate, and the
like. The prefix "acyl" as applied to a phosphoglyceryl refers to a
carbonyl-containing moiety, such as RC(O)--.
[0133] Presently preferred groups having a formula RO-- include but
are not limited to hydroxy, methoxy, ethoxy, phenoxy, benzyloxy,
1-O-alkyl-2-O-sn-glyceroyl 3-O-phosphate (or
2-O-alkyl-1-O-sn-glyceroyl 3-O-phosphate),
1-O-alkenyl-2-O-sn-glyceroyl 3-O-phosphate (or
2-O-alkenyl-1-O-sn-glyceroyl 3-O-phosphate),
1-O-acyl-2-O-sn-glyceroyl 3-O-phosphate (or
2-O-acyl-1-O-sn-glyceroyl 3-O-phosphate), in which the phosphate
group may be esterified (with an organic base, an alcohol, or a
sugar), and the like.
[0134] Presently preferred groups having a formula RC(O)-- for use
in accordance with the present invention, include but are not
limited to acetyl, formyl, benzoyl, and the like.
[0135] Presently preferred groups having a formula RC(O)O-- for use
in accordance with the present invention include but are not
limited to formyloxy, acetoxy, and the like.
[0136] Presently preferred groups having a formula ROC(O)-- for use
in accordance with the present invention include but are not
limited to methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl,
benzyloxycarbonyl, and substituted phosphoglyceryls wherein R is
selected from 1-O-alkyl-2-O-sn-glyceroyl 3-O-phosphate,
2-O-alkyl-1-O-sn-glyceroyl 3-O-phosphate,
1-O-alkenyl-2-O-sn-glyceroyl 3-O-phosphate,
2-O-alkenyl-1-O-sn-glyceroyl 3-O-phosphate,
1-O-acyl-2-O-sn-glyceroyl 3-O-phosphate, 2-O-acyl-1-O-sn-glyceroyl
3-O-phosphate (in each of which the phosphate group may be
esterified with an organic base, an alcohol, or a sugar), and the
like.
[0137] Presently preferred groups having a formula (R).sub.2N-- for
use in accordance with the present invention include but are not
limited to amino, methylamino, ethylamino, phenylamino,
dimethylamino, diethylamino, and the like.
[0138] Presently preferred groups having a formula RC(O)N-- for use
in accordance with the present invention include but are not
limited to acetylamino, benzoylamino, and the like.
[0139] Presently preferred groups having a formula R(NH)C(O)-- for
use in accordance with the present invention include but are not
limited to substituted phosphoglyceryls wherein R is selected from
1-O-alkyl-2-N-iminoglyceryl 3-O-phosphate,
2-O-alkyl-1-N-iminoglyceryl 3-O-phosphate,
1-O-alkenyl-2-N-iminoglyceryl 3-O-phosphate,
2-O-alkenyl-1-N-iminoglyceryl 3-O-phosphate,
1-O-acyl-2-N-iminoglyceryl 3-O-phosphate,
2-O-acyl-1-N-iminoglyceryl 3-O-phosphate (in each of which the
phosphate group may be esterified with an organic base, an alcohol,
or a sugar), and the like.
[0140] A subset of groups of the preceding formula includes
functionalities wherein R is 1-alkenyl-1-hydroxy-2-N-iminoglyceryl
3-O-phosphate, with tetradeca-1-ene-1-yl being a presently
preferred alkenyl and the phosphate group being optionally
esterified with an organic base, an alcohol or a sugar. This subset
corresponds to sphingolipid derivatives, as shown below (glycerol
backbone is drawn bold):
##STR00018##
[0141] A presently preferred group having a formula RN.dbd.N-- for
use in accordance with the present invention includes but is not
limited to phenylazo, and the like.
[0142] Presently preferred groups having a formula RS-- for use in
accordance with the present invention include but are not limited
to ethylthio, S-cysteinyl, S-glutathionyl,
1-O-alkyl-2-S-thiaglyceryl 3-O-phosphate (or
2-O-alkyl-1-S-thiaglyceryl 3-O-phosphate),
1-O-alkenyl-2-S-thiaglyceryl 3-O-phosphate (or
2-O-alkenyl-1-S-thiaglyceryl 3-O-phosphate),
1-O-acyl-2-S-thiaglyceryl 3-O-phosphate (or
2-O-acyl-1-S-thiaglyceryl 3-O-phosphate), in which the phosphate
group may be esterified (with an organic base, an alcohol, or a
sugar), and the like.
[0143] A presently preferred group having a formula RSO.sub.2-- for
use in accordance with the present invention includes but is not
limited to methylsulfonyl, benzylsulfonyl, and the like.
[0144] A presently preferred group having a formula RS(O)-- for use
in accordance with the present invention includes but is not
limited to methylsulfinyl, benzylsulfinyl, and the like.
[0145] A presently preferred group having a formula RSO.sub.2O--
for use in accordance with the present invention includes but is
not limited to methylsulfonyloxy, benzylsulfonyloxy, and the
like.
[0146] A presently preferred group having a formula RS(O)O-- for
use in accordance with the present invention includes but is not
limited to methylsulfinyloxy, benzylsulfinyloxy, and the like.
[0147] Presently preferred groups having a formula RSC(O)-- for use
in accordance with the present invention include but are not
limited to 1-O-alkyl-2-S-thiaglyceryl 3-O-phosphate (or
2-O-alkyl-1-S-thiaglyceryl 3-O-phosphate),
1-O-alkenyl-2-S-thiaglyceryl 3-O-phosphate (or
2-O-alkenyl-1-S-thiaglyceryl 3-0-phosphate),
1-O-acyl-2-S-thiaglyceryl 3-O-phosphate (or
2-O-acyl-1-S-thiaglyceryl 3-O-phosphate), in which the phosphate
group may be esterified (with an organic base, an alcohol, or a
sugar), and the like.
[0148] It has been found that
Z(-)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide is at least four
times more active as an integrin modulator than other enantiomers,
racemic E-ajoene, or the unseparated 3:1 mixture of racemic E and
Z-ajoenes. The superior activity of one out of the four enantiomers
correlates with the presence in the molecule of the following
substructure:
##STR00019##
[0149] Chiral compounds containing the above-described core
structure thus comprise a class of potent integrin modulators.
Specifically, this class includes compounds of the formula:
##STR00020##
The formula (2) above corresponds to ajoene when X.sup.1 and
X.sup.2 are both CH.sub.2.dbd.CH--CH.sub.2--, m is 1, and n is
0.
[0150] A mixture of four enantiomers, including a compound of
formula (2) as one of the enantiomers, can be synthesized by
methods well known in the art, such as those described in U.S. Pat.
Nos. 4,643,994 and 4,665,088, in Block et al., J Am. Chem. Soc.,
1986, 108, 7045, and in Sendl et al., Planta Med., 1991, 57, 361.
The entire contents of all four documents are incorporated herein
by reference, except that in the event of any inconsistent
disclosure or definition from the present application, the
disclosure or definition herein shall be deemed to prevail. Such a
mixture, having a general formula (2a), can then be separated as
described below to obtain a desired compound, for example,
(Z)-(-)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide:
##STR00021##
As further described below, compounds of formula (2a) may be
cleaved at the disulfide bond and converted to DDCs in accordance
with the present invention.
[0151] U.S. Pat. No. 4,665,088 describes the synthesis of
(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide. Briefly, garlic is
subjected to any convenient extraction procedure which acts to
isolate the allicin component of garlic so that this component can
be thereafter dissolved in an appropriate lower alkanol for a time
and at a temperature sufficient to form
(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide. To obtain higher
yields, the garlic should be freshly cut, chopped or ground. Whole
garlic cloves reduce yield, but can be satisfactorily used. The
garlic pieces are blended with a volatile, water-miscible organic
solvent such as a lower alkanol, ether, or acetone and allowed to
sit for several hours or days. The particulate material is usually
removed prior to further processing. Vacuum concentration of the
liquid and extraction of the aqueous residue with an appropriate
solvent, such as diethyl ether, appears to increase the yield
significantly. The extracted aqueous residue can be washed several
times with water, dried and evaporated to increase the purity of
the oily allicin residue. The oily residue product is then
dissolved in a volatile organic solvent, such as acetone or a lower
alkanol in mixture with water (10-90%), and maintained at a
temperature of from about -40.degree. C. to a temperature less than
about the reflux temperature of the organic solvent in mixture with
the water. Generally, the higher the temperature, the lower the
amount of time the mixture should be maintained at that
temperature. It is generally desirable to adjust temperature to
achieve a maintenance time of several hours, usually from about
10-72 hours. (E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide can
also be prepared as described below.
[0152] U.S. Pat. No. 4,643,994 describes the synthesis of compounds
of formula (2a)--that is, mixtures of enantiomers, which contain
several compounds that fall within formula (2). This patent also
sets forth a general synthetic method that can be used for
preparing compounds of formula (2a). Briefly, an appropriate
disulfide radical having the formula:
X.sup.1--S--S-Q (3)
wherein Q is --(CH.sub.2).sub.m--CH.dbd.CH--(CH.sub.2).sub.n--H,
and m, n, and X.sup.1, may be as defined above for the DDCs
embodying features of the present invention, is treated with an
oxidizing agent, preferably in the presence of a solvent, and
preferably at a temperature of from about -40.degree. C. to about
65.degree. C. to produce a thiosulfinate of the formula:
##STR00022##
[0153] The thiosulfinate is then heated, typically refluxed, in the
presence of an appropriate solvent, preferably a 60:40 organic
solvent:water mixture to form a trithio oxide of the formula:
##STR00023##
[0154] Typically, the reaction which causes the formation of the
trithio oxide of formula (5) also causes the formation of minor
products wherein each X.sup.1 or Q can be Q or X.sup.1,
respectively. If a mixture of disulfides or thiosufinates is used
as the starting compounds, the product will be a further mixture of
products. The mixture of products can be separated at this point in
the process by various means, such as extraction, or the mixture
can be maintained as such through the next step(s). The compound
(5) can be used directly to make DDCs or can be further oxidized to
produce additional compounds of formula (2). Treatment with a
stoichiometric amount (or a slight excess) of oxidizing agent forms
compounds of formula:
##STR00024##
[0155] Treatment with further oxidizing agents at -30.degree. C. to
40.degree. C. produces compounds of formula:
##STR00025##
[0156] Continued treatment with an oxidizing agent produces
compounds of formula:
##STR00026##
[0157] Each of compounds (7) and (8) can be reacted with a thiol of
the formula X.sub.1SH, or an alkali metal salt thereof, wherein
X.sub.1 is as defined above, to produce further compounds having
different selected substituents as the X.sub.1 moiety. These
compounds can be purified by various means, including extraction,
and used to make DDCs. The DDCs can be purified from an isomeric
mixture by high performance liquid chromatography (HPLC) on columns
packed with sorbents that are capable of separating individual
enantiomers from mixtures thereof (cf. Examples below).
[0158] As described above, the integrin-modulating activities of
ajoene, including the activity of Z(-)-ajoene as a CIM, may be
traced to the ability of ajoene and its enantiomers to form in vivo
enantiomeric (2E)-(+)-, (2E)-(-)-, (2Z)-(+)-, and (2Z)-(-)-DODOyl
substituted compounds of the formula:
##STR00027##
wherein X.sup.2 may correspond to a free radical or a moiety
attached thereto.
[0159] Thus, DDCs embodying features of the present invention
include derivatives of compounds of formula (9), and have a general
formula:
##STR00028##
wherein X.sup.1 and X.sup.2 are as defined above.
[0160] Compounds of formula (10) may be accessed from compounds of
formula (2a) by various methodologies well known in the art. For
example, the disulfide bond of (2a) may be cleaved with a mild
reducing agent (e.g., zinc and dilute acid; Ph.sub.3P and H.sub.2O;
heating with alkali; etc.) to form the two corresponding thiols.
The DDC fragment (i.e., formula (10) in which X.sup.2 is hydrogen)
may be elaborated to introduce various other X.sup.2 groups to form
DDCs in accordance with the present invention (e.g., by
nucleophilic substitution reactions known in the art, wherein a
leaving group in the X.sup.2 residue to be attached is displaced by
the thiol sulfur atom, in the DDC fragment, or a salt thereof).
[0161] Presently, a particularly preferred DDC subgroup is
constituted by the prototypal DODOyl substituted compounds of
formula (9)--that is, compounds of formula (10), wherein
X.sup.1=allyl, m=1, and n=0.
[0162] Yet another distinct DDC subgroup includes compounds which
share two common characteristics: (a) the polymeric nature of
X.sup.2 and (b) multiplicity of the active chiral moieties attached
to a single X.sup.2. These compounds have a general formula:
##STR00029##
[0163] wherein X.sup.2 is a polysaccharide, polypeptide, or other
suitable substrate;
[0164] X.sup.1, m, and n are the same as defined for formula (10)
above; and w is an integer from 7 to 1000.
[0165] A compound of formula (11), wherein X.sup.2 is a
polysaccharide, is a polymer of furanose and/or pyranose units
carrying N-, C-, or S-linked DDC functions, or derivatives
thereof.
[0166] A compound of formula (11), wherein X.sup.2 is a
polypeptide, is a synthetic structure the cysteine residues of
which carry S-linked DDC functions, or derivatives thereof.
[0167] Polymeric DDCs may also have the formula:
##STR00030##
[0168] wherein X.sup.1 and X.sup.2 may be different and are
preferentially selected from functionalities enabling introduction
of the chiral moiety into a phospholipid, glycolipid, or
sphingolipid (e.g., allyl, allylthio, substituted furanose/pyranose
units, polycyclic aryls, and substituted phosphoglyceryls), with
subsequent incorporation of these lipids into liposomes or
micelles.
[0169] A typical compound of formula (12), therefore, is a
liposomal preparation containing a lipid modified, via
S-alkylation, N-alkylation, O-alkylation, or O-acylation, with a
DDC function or its derivative.
[0170] Polymeric DDCs may also have the formula:
##STR00031##
[0171] wherein X.sup.1 and X.sup.2 are the same or different and
comprise at least one functional group configured for participation
in a polymerization reaction;
[0172] wherein m is an integer from 0 to 30;
[0173] wherein n is an integer from 0 to 30; and
[0174] wherein w is an integer from 2 to 1000.
[0175] Polymeric DDCs may also have the formula:
##STR00032##
[0176] wherein X.sup.1 comprises at least one functional group
configured for participation in a polymerization reaction;
[0177] wherein m is an integer from 0 to 30;
[0178] wherein n is an integer from 0 to 30; and
[0179] wherein w is an integer from 2 to 1000.
[0180] It is understood that any DDC of formulae (10)-(12), (35),
and (40) includes the E and the Z stereoisomers (generated by the
geometry of the central double bond), each having the R and the S
configurations (generated by the presence of the central sulfoxide
functionality). It is appreciated that, in one illustrative aspect,
the compounds described herein can be mixtures containing all four
enantiomers--that is, (E,R), (E,S), (Z,R), and (Z,S). In another
illustrative aspect, the compounds described herein can be mixtures
of two enantiomers having one double bond geometry or the other
that is, (E,R) and (E,S) or (Z,R) and (Z,S). In another
illustrative aspect, the compounds described herein can be mixtures
of two stereoisomers having the same sulfoxide chirality that is,
(E,R) and (Z,R) or (E,S) and (Z,S). In another illustrative aspect,
the compounds described herein can be single enantiomers having one
sulfoxide chirality and one double bond geometry that is, (E,R),
(E,S), (Z,R) or (Z,S). In another illustrative aspect, the
compounds described herein can be mixtures of two enantiomers not
matching each other in both the double bond geometry and the
sulfoxide chirality that is, (E,R) and (Z,S) or (E,S) and (Z,R). In
yet another illustrative aspect, the compounds can be mixtures of
any three out of the four possible enantiomers.
[0181] In accordance with the present invention, safe and effective
doses of the DDCs may inhibit the progression of an HIV infection
in a patient or the infection of an uninfected patient by HIV.
While Z(-)-ajoene is one such inhibitor, other DDCs may also be
used.
[0182] One of the characteristics of the inhibition of HIV
infection is the diminution of the formation of HIV-induced
syncytia, in which HIV target cells, such as lymphocytes and
monocytes, fuse together to form giant, multinucleate cells.
Transfer of genetic material between cells may, thereby, also be
inhibited by DDCs, which inhibit fusion with cell membranes.
[0183] Additionally, because DDCs may modulate pertinent
integrin-mediated activity, DDCs may inhibit the entry of the
infective HIV material into its target cells, including
CD4-negative cells, both virus-to-cell and cell-to-cell entry, and
the production of HIV and other viruses by the infected cells. For
these purposes, DDCs in accordance with the present invention may
preferably be administered in a sufficient dose to provide a
concentration approaching or exceeding 5 micromoles per liter of a
patient's blood plasma, although lesser concentrations may also be
effective and may also be effective in sustained (i.e., chronic)
administration. This dose may be effective when the DDCs are used
alone or in bi- or multi-therapy addressing different disease
parameters (e.g., the function of viral enzymes).
[0184] In addition to infections caused by HIV and other viruses of
the Retroviridae family, DDCs embodying features of the present
invention may be used locally or systemically to inhibit or prevent
the transmission, in vivo and in vitro, of other viruses infecting
humans and other animals. In particular, DDCs embodying features of
the present invention may be used to treat or prevent infections
caused by viruses, the transmission of which involves fusion of at
least a part of the virus with the membrane of the target cell that
is to be infected. Such viruses include all enveloped viruses and
other viruses that infect cells in this manner. The enveloped
viruses include the Retroviridae, Herpesviridae (e.g., herpes
simplex virus 1 [HSV-1], HSV-2, varicella zoster, Epstein-Barr
virus, and cytomegaly virus), Hepadnaviridae (e.g., hepatitis B),
Flaviviridae (e.g., yellow fever virus and hepatitis C virus),
Togaviridae (e.g., rubivirus, such as rubella virus, and
alphavirus), Orthomyxoviridae (e.g., influenza virus),
Paramyxoviridae (e.g., measles, parainfluenza, mumps and canine
distemper viruses), Poxviridae (e.g., variola virus and vaccinia
virus), and Rhabdoviridae (e.g., rabies virus). Other viruses that
infect cells by fusing with the membrane include Papovaviridae
(e.g., papillomavirus), Picornaviridae (e.g., hepatitis A virus and
poliomyelitis virus), Rotaviridae, and Adenoviridae. In addition to
inhibiting or preventing virus-to-cell entry, DDCs may also inhibit
or prevent cell-to-cell transmission of viruses (e.g., by
inhibiting or preventing syncytia formation or by inhibiting or
preventing intercellular viral transfer between cells in contact or
close proximity) and the production of viruses by infected
cells.
[0185] DDCs embodying features of the present invention may also
serve as agents that inhibit the adhesion, migration (e.g.,
chemotaxis or other infiltration of the tissue), and aggregation of
various cell types and lines, including blood platelets and
neutrophils. Thus, DDCs in accordance with the present invention
may exhibit benefit as agents for the treatment of pathologies
derived from adhesion, migration, and aggregation of these and
other cells, including thrombosis and various types of
inflammation.
[0186] Thrombosis is defined as blockage of blood vessel(s) by
thrombi (i.e., clots formed from fibrin and platelet aggregates)
deposited on the inner surface of the vessel. Thrombi form in
arteries (e.g., damaged as a result of a disease) or in veins
(e.g., due to lengthy immobilization). If a thrombus or a blood
clot is dislodged and moves through the bloodstream to create an
obstruction outside the place of its formation, it becomes an
embolus (hence the terms "thromboembolism" and "thromboembolic
disease"). Thrombosis or thromboembolism of coronary arteries can
cause heart attacks and myocardial infarction; the same processes
in brain arteries cause stroke. Inhibition of platelet aggregation
by DDCs in accordance with the present invention may, therefore,
arrest thrombosis at early stages, precluding the development of
thrombotic and thromboembolic diseases.
[0187] Inflammation, a pathological process inherent in a variety
of distinct diseases and illnesses, is defensive in nature, but
potentially dangerous if uncontrolled. When viewed at the "whole
body" level, an inflammation is most frequently characterized by
several localized manifestations (indices), including hemodynamic
disorders (e.g., hyperemia and edema), pain, temperature increment,
and functional lesion. These inflammatory phenomena are underlain
by events at the cellular and molecular levels. At the cellular
level, inflammation is characterized by leukocyte extravasation (a
process involving adhesion of leukocytes to the endothelium of the
vessel wall and migration into tissue where they may phagocytose
bacteria, viruses, and cell debris) and platelet aggregation (a
mechanism, inter alia, whereby the spread of the infection is
prevented). At the molecular level, inflammation is characterized
by activation of at least three plasma defense systems (complement,
kinin, and coagulation/fibrinolysis cascades) and by synthesis of
cytokines and eicosanoids. When inflammation becomes generalized
(e.g., as in the case of shock), various indices of inflammation
occur systemically throughout the entire organ/organism. In cases
of shock, platelets and leukocytes (principally neutrophils)
aggregate in the blood vessels, leading to the development of a
clinical condition known as multiple organ failure. The primary
organ affected in shock patients is commonly the lung. Lung
failure, or adult respiratory distress syndrome (ARDS), a
destructive inflammation resulting from adhesion, aggregation, and
degranulation of activated neutrophils in the pulmonary
microvasculature, may be the main cause of death in patients
suffering shock. DDCs embodying features of the present invention
may thus counteract at least part of the effects of shock, whether
arising, for example, from sepsis, anaphylaxis, blood loss, or from
other precipitating events.
[0188] DDCs embodying features of the present invention may also be
administered in effective dosages to suppress many other acute
inflammatory processes, such as those associated with peritonitis,
meningitis, and ischemia-reperfusion. Ischemia-reperfusion injury
occurs (e.g., in heart, brain, kidney, liver, lung, intestinal
tract, or any limb) when blood supply is abruptly stopped
(ischemia) and then resumed (reperfusion) after a short period.
[0189] With the onset of ischemia and the decrease in the perfusion
pressure, neutrophils are retained in the capillaries. As the
ischemia progresses, cytokines (and other chemoattractants) are
released into the capillary lumina in regions of the tissue where
the blood flow blockage has occurred, increasing the adhesiveness
of the retained neutrophils to the endothelium and to each other.
Aggregates of neutrophils thus formed obstruct postcapillary
venules ("no-reflow" or "no-washout") and attenuate the restoration
of the blood flow in the affected region, precluding its
reoxygenation and extending the area of ischemia. Activated
neutrophils trapped in the capillaries also release hydrolytic
enzymes and reactive oxygen species (i.e., the armamentarium
ordinarily used to defend the host against microorganisms),
producing a destructive inflammation.
[0190] Restoration of the blood flow, however, further augments the
severity of the inflammation thus developed. Neutrophils arriving
to the previously ischemic region are activated (by
chemoattractants and/or products released by the trapped
neutrophils) and recruited into the tissue, where the defensive
machinery of the cells is once again used against the host
(secondary injury). Ischemia-reperfusion injury can also be
generalized, for example, in the case of resuscitation after
hemorrhagic shock (A. Mazzone et al., "Leukocyte CD 11/CD18
Integrins: Biological and Clinical Relevance," Haematologica 1995,
80, 161; W. H. Reinhart, "Hemorheology: Blood Flow Hematology,"
Schweiz. Med. Wochenschr. 1995, 125, 387).
[0191] DDCs embodying features of the present invention may be
potent inhibitors of adhesive interactions for other cells, such as
lymphoid cells. Adhesion of lymphocytes to each other and to
nonlymphoid cells is prerequisite to the development of any immune
response. Thus, DDCs in accordance with the present invention may
serve as agents for the prevention, treatment, and control of
adverse, undesirable, and self-destructive immune responses.
[0192] One group of such immunopathologies includes diseases
stemming from divers allergic reactions (e.g., delayed type
hypersensitivity, Arthus reaction, and anaphylaxis). Allergy is an
anomalous immune response to antigen challenge, characterized by
recruitment of specific leukocyte subsets (e.g., cytotoxic
lymphocytes and/or eosinophils) to the tissue, resulting in
inflammation. Development of allergic inflammation is the main
component in the pathogenesis of many diseases and illnesses,
including, for example, asthma, eczema, purpura pigmentosa
chronica, various vasculitides, and hay fever, in addition to those
described above. DDCs in accordance with the present invention may
serve to control these diseases and illnesses.
[0193] Allograft rejection is another example of an undesirable
immune response, in which the transplanted organ is recognized by
the immune system as a foreign body ("non-self") and attacked in
sequence by cytotoxic lymphocytes and phagocytes recruited from the
circulation. This inflammatory response results in progressive
disruption of the tissue, including graft necrosis. DDCs embodying
features of the present invention may be used to prevent cell
recruitment into transplanted tissue and thereby prolong graft
survival by reducing both acute and chronic aspects of
rejection.
[0194] Moreover, the transplanted organ also contains lymphocytes,
which, in turn, recognize their new environment as "non-self." The
immune response initiated by these donor lymphocytes in the body of
the recipient produces a condition known as graft-versus-host
disease (GVHD), which can lead to injury, both acute and chronic.
DDCs embodying features of the present invention may contribute to
the control of both acute and chronic GVHD.
[0195] Any method of treatment that suppresses both rejection and
susceptibility to viruses (which, like cytomegaly virus, frequently
contaminate the transplanted organs and decrease the probability of
their engraftment) will have an extra benefit to the graft
recipient. As described above, DDCs embodying features of the
present invention may exert pronounced antiviral effects, in
addition to being potent anti-inflammatory agents. Thus,
administration of DDCs to patients undergoing organ transplantation
offers much promise as a novel therapeutic approach to the
prevention of rejection. Self-destructive responses are caused by
the failure of the immune system to distinguish "self" from
"non-self." This group of immunopathologies includes a wide variety
of diseases (herein termed collectively "autoimmune diseases"),
including but not limited to rheumatoid arthritis, systemic lupus
erythematosus, Sjogren's syndrome, multiple sclerosis,
insulin-dependent diabetes mellitus, glomerulonephritis, Graves
disease, Hashimoto's thyroiditis, and vasculitides. Other
conditions and diseases may also fall into this category (e.g., see
the description of psoriasis below) or include a component that
does so (e.g., chronic viral diseases stimulating an autoimmune
response). In spite of pronounced differences in the clinical
picture of the various autoimmune diseases, the underlying
mechanisms involve, in every case, undesirable recruitment of
leukocytes to organs/tissues affected, resulting in destructive
inflammation. DDCs embodying features of the present invention may
be used to reduce or prevent this cellular recruitment and thereby
suppress the abnormal immune response. Accordingly, DDCs in
accordance with the present invention may be used to treat
autoimmune diseases.
[0196] Without wishing to be bound to a particular theory or in any
way limit the scope of the appended claims or their equivalents, it
is presently believed that the beneficial effects of DDCs in
accordance with the present invention are achieved because these
substances are modulating integrin-mediated functions. As used
herein, "modulate" means to affect the development or expression of
modalities normally characterizing cellular activities and/or
functions mediated by integrins. A modulating agent may act on an
integrin directly, for example, by binding to or interacting with a
portion of at least one subunit (alpha or beta) of the integrin.
The agent may also act in some other fashion that is not considered
direct, for example, through any of the various cellular substances
and structures which ordinarily interact with or enable the
participation of specific integrins, alone or in combination. These
substances and structures include but are not limited to
transmembrane proteins (e.g., integrins themselves and
integrin-associated proteins), membrane phospholipids,
intracellular molecules with messenger-like function (e.g.,
integrin-modulating factor), enzymes, lipid rafts, and regulatory
and signaling proteins. Thus, for example, a modulation may result
from alteration in integrin expression, activation, conformation,
association or disassociation of the alpha and beta integrin
subunits (or any parts thereof) affecting integrin clustering or
clustering abilities, association or disassociation of integrin
clusters (and of clusters formed by integrins with other proteins),
or from the variations of integrin-cytoskeleton associations,
although modulation may also occur from other types of effects. The
functions of integrins, as defined herein, are interrelated and
include but are not limited to, inter alia, signaling, adhesion,
fusion, internalization, cellular conformation, regulation of lipid
raft distribution, and microtubule stabilization (For recent
research vis-a-vis several of these functions, see: J-L Guan,
"Integrins, Rafts, Rac, and Rho," Science, 2004, 303, 773-774; A.
F. Palazzo et al., "Localized Stabilization of Microtubules by
Integrin- and FAK-Facilitated Rho Signaling," Science, 2004, 303,
836-839; and M. A. Del Pozo et al., "Integrins Regulate Rac
Targeting by Internalization of Membrane Domains," Science, 2004,
303, 839-842).
[0197] As a result of their ability to modulate activities in which
integrins participate, DDCs embodying features of the present
invention may be used to treat a plurality of diseases or
conditions that involve undesirable integrin-mediated functions as
a mechanism, including those described above. For instance, DDCs in
accordance with the present invention may be used to inhibit
virus-cell fusion, to modulate molecular exchanges arising from
cell-to-cell cytoplasmic interactions, and/or to otherwise modulate
undesired cell-cell fusion.
[0198] Undesired cell-cell fusion may include, for example,
cell-cell fusion (transitory or permanent) that results in the
transfer of viral genetic material; cell-cell fusion that results
in the formation of multinucleate cells (e.g., syncytia, giant
cells, and osteoclasts); undesired fertilization of eggs by sperm;
and the formation of multinucleate germinal cells
(syncytiotrophoblast).
[0199] Thus, DDCs embodying features of the present invention may
be used as contraceptives, being administered per vaginam
(topically), per os, or in any other appropriate way, when used for
this purpose. DDCs in accordance with the present invention may
also prevent conception, however, at the stage of embryo
implantation. For example, DDCs may prevent the initial adhesion of
the blastocyst to the endometrium and the migration of
cytotrophoblasts through the maternal epithelium (i.e., processes
similar to certain steps in the leukocyte extravasation cascade and
tumor cell metastasis).
[0200] Furthermore, DDCs embodying features of the present
invention may be capable of inhibiting cytotrophoblast invasion, a
process differing from extravasation in that it goes from the
tissues to the vascular lumen and in that the invading cells cross
the blood-tissue barrier from outside of the vessel (reverse
diapedesis). For example, the production of proteolytic enzymes
that are used by cytotrophoblasts to penetrate the basement
membrane is governed by integrin outside-in signaling. Modulation
of the signaling function of integrins by DDCs in accordance with
the present invention may either completely prevent the production
of the requisite enzymes or attenuate it to an extent precluding
invasion. A related mechanism underlies the ability of DDCs to
block angiogenesis, preventing the blood supply to the fetal
tissue. Thus, DDCs embodying features of the present invention may
be used as effective emergency contraceptives to prevent unwanted
pregnancy or to interrupt it at an early stage. There are
additional mechanisms whereby DDCs, when desired, can exert
contraceptive effects. For example, they can prevent the
chemotactic response of sperm in the vaginal environment (a
specific case of cell homing) and sperm interactions with the
epithelium of the female genital tract. Moreover, DDCs embodying
features of the present invention may be administered to males to
modulate integrin-mediated functions in sperm precursors and other
testicular or epididymal cells, thereby interfering with the
maturation processes and resulting in the production of
fertilization-incompetent gametes or inhibition of
fertilization-competent gametes.
[0201] The development of major bone diseases, including
osteoporosis, is underlain by excessive bone resorption. This
fundamental function is performed by osteoclasts. Osteoclasts are
multinucleate bone cells formed by fusion of mononuclear
progenitors called preosteoclasts. The regulation of osteoclast
formation may be achieved by agents acting at various levels of
osteoclast formation, including preosteoclast fusion (Zaidi et al.,
"Cellular Biology of Bone Resorption," Biol. Rev. 1993, 68, 197).
DDCs embodying features of the present invention may regulate bone
resorption because they inhibit the fusion of preosteoclasts,
necessary for the formation of osteoclasts.
[0202] Granulomas are characteristic of chronic inflammatory
lesions, such as those found in tuberculosis and other chronic
infections. Granulomas are also present in sarcoidosis, a chronic,
systemic inflammatory disease of unclear etiology. Granulomas
present in cases of chronic infection and in sarcoidosis contain a
large number of multinucleate giant cells formed by the fusion of
macrophages. Other diseases associated with the formation of
multinucleate cells include but are not limited to Crohn's disease,
Langerhans cell histiocytosis, and giant cell arteriitis. DDCs
embodying features of the present invention may be used to inhibit
the formation of these giant multinucleate cells with beneficial
therapeutic effects.
[0203] Excessive formation of fibrous interstitial tissue (i.e.,
fibrosis, or sclerosis including, for example, multiple sclerosis
and scleroses of specific organs, such as sclerosis of the liver)
is characteristic of certain diseases (such as scleroderma and
idiopathic pulmonary fibrosis) and an outcome of chronic
inflammatory processes (e.g., glomerular fibrosis). The development
of fibrotic lesions and progression of fibrosis, associated with
these conditions, diseases, and illnesses, has been linked to
abnormal integrin expression and altered cell adhesion patterns.
DDCs embodying features of the present invention may, therefore, be
used for treatment of fibrotic lesions, including the formation of
keloid (scar tissue). Lesions observed in skin diseases and
illnesses of diverse origin, such as lichen planus, urticaria,
dermatofibroma, psoriasiform dermatitides, and keratoses, are
characterized by aberrant integrin expression. DDCs in accordance
with the present invention may serve as agents for the symptomatic
treatment of these diseases, being administered topically,
intradermally, and subcutaneously at the site of lesions, or in any
other appropriate way, when used for this purpose.
[0204] Another disease characterized by the formation of cutaneous
lesions is psoriasis. Although the etiology of psoriasis attends
further elucidation (several viruses and an autoimmune component
could be involved), its pathogenesis is associated with abnormal
expression of integrins in target tissue (e.g., in vascular cells,
keratinocytes, and dendritic cells), proliferation of endothelial
and epidermal cells, and an autoimmune component (recruitment of
lymphocytes and macrophages to skin and joints). DDCs embodying
features of the present invention may, therefore, be used to treat
psoriasis in multiple respects.
[0205] As a result of their antiviral and anti-inflammatory
activity, DDCs embodying features of the present invention may
exhibit significant potency in the prevention and treatment of
certain diseases with combined etiopathogenesis. As roughly
elaborated herein, the term "etiopathogenesis" is used in reference
to diseases for which no distinction can be drawn thus far as to
etiology and pathogenesis. In addition to psoriasis, described
above, a good example of such a disease is atherosclerosis. A
variety of pathogens may participate in the development of
atherosclerosis. One of the best studied viral contributors is
cytomegalovirus, which induces a specific type of infection
characterized by plaque formation along the blood vessels (J. L.
Melnick et al., "Cytomegalovirus and Atherosclerosis," BioEssays
1995, 17, 899). A prominent feature of atherosclerosis is the
recruitment of monocyte-macrophages into atherosclerotic plaques,
which is an integrin-mediated process. Also, proliferation of
smooth muscle cells, which contributes to the formation of
atherosclerotic lesions, is regulated by integrins. As described
above, DDCs embodying features of the present invention may inhibit
the transmission of viral infections virus-to-cell and
cell-to-cell. Integrin-mediated adhesion and signaling may also be
inhibited by DDCs. Thus, for multiple reasons, DDCs in accordance
with the present invention may be used to treat diseases involving
combinations of integrin-mediated etiopathogenesic factors,
including those that are in part of viral nature.
[0206] Certain neurodegenerative disorders of unclear etiology
(e.g., Alzheimer's disease and amyotrophic lateral sclerosis)
involve autoimmune inflammation of nervous tissue as a pathogenetic
mechanism. Thus, DDCs embodying features of the present invention
may demonstrate significant potency in mitigating the symptoms of
these diseases and slowing their progression. This conclusion is
further supported by various studies which show that other
anti-inflammatory treatments benefit Alzheimer's patients (P. L.
McGeer et al., "The Inflammatory Response System of Brain:
Implications for Therapy of Alzheimer's and Other Neurodegenerative
Diseases," Brain Res. Brain Res. Rev. 1995, 21, 195; J. C. Breitner
et al., "Delayed Onset of Alzheimer's Disease with Nonsteroidal
Anti-Inflammatory and Histamine H.sub.2 Blocking Drugs," Neurobiol.
Aging 1995, 16, 523).
[0207] To treat or prevent any of these disorders, diseases or
conditions, an effective amount of a DDC embodying features of the
present invention may be administered to an animal in need thereof.
Preferably, the animal is a mammal, such as a rabbit, goat, dog,
cat, horse or human. Effective dosage forms, modes of
administration, and dosage amounts may be determined empirically,
and making such determinations lies well within the skill of the
ordinary artisan. It is understood by those of ordinary skill in
the art that the dosage amount will vary with the disorder, disease
or condition to be treated or prevented, the severity of the
disorder, disease or other condition, which integrin-mediated
function(s) is (are) to be modulated, the route of administration,
the rate of excretion, the duration of the treatment, the identity
of any other drugs being administered, the age, size and species of
animal, and like factors well known in the arts of medicine and
veterinary medicine. Typically, a suitable daily dose of a DDC will
be that amount of the compound which is the lowest dose effective
to produce the desired effect. The effective daily dose of a DDC
embodying features of the present invention may be administered as
two, three, four, five, six or more sub-doses, administered
separately at appropriate intervals throughout the day. An existing
disorder, disease or condition treated with a DDC or combination of
DDCs in accordance with the present invention may be reduced,
inhibited, suppressed or eliminated or one or more symptoms of the
disorder, disease or condition may be alleviated or eliminated.
[0208] DDCs embodying features of the present invention may be
administered in any desired and effective manner: as pharmaceutical
compositions for oral ingestion, or for parenteral or other
administration in any appropriate manner such as intraperitoneal,
subcutaneous, topical, intradermal, inhalation, intrapulmonary,
rectal, vaginal, sublingual, intramuscular, intravenous,
intraarterial, intrathecal, or intralymphatic. For example, the
topical application of DDCs to mucous membranes (in the form of
creams, gels, suppositories, and other known means of topical
administration) may be used to prevent HIV infection of mucosal
cells, an important route of HIV transmission. In addition,
intralymphatic administration of DDCs may be advantageous in
preventing the spread of HIV within the body. Further, DDCs in
accordance with the present invention may be administered in
conjunction with other treatments for the disorder, disease or
condition being treated with the DDC, such as other antiviral
drugs, other contraceptives, and other anti-shock or
anti-inflammatory drugs or treatments. DDCs in accordance with the
present invention may be encapsulated or otherwise protected,
against gastric or other secretions, if desired.
[0209] While it is possible for a DDC embodying features of the
present invention to be administered alone, it is presently
preferred that the DDC be administered as a pharmaceutical
formulation (composition). The pharmaceutical compositions in
accordance with the present invention may include one or more DDCs
as an active ingredient in admixture with one or more
pharmaceutically-acceptable carriers and, optionally, one or more
other compounds, drugs, ingredients and/or materials. Regardless of
the route of administration selected, the DDCs embodying features
of the present invention may be formulated into
pharmaceutically-acceptable dosage forms by conventional methods
known to those of ordinary skill in the art (e.g., see: Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.).
Pharmaceutical carriers are well known in the art (e.g., see:
Remington's Pharmaceutical Sciences cited above and The National
Formulary, American Pharmaceutical Association, Washington, D.C.)
and include sugars (e.g., lactose, sucrose, mannitol, and
sorbitol), starches, cellulose preparations, calcium phosphates
(e.g., dicalcium phosphate, tricalcium phosphate and calcium
hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g.,
saline, sodium chloride injection, Ringer's injection, dextrose
injection, dextrose and sodium chloride injection, lactated
Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol,
and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and
polyethylene glycol), organic esters (e.g., ethyl oleate and
triglycerides), biodegradable polymers (e.g.,
polylactide-polyglycolide, poly[orthoesters], and
poly[anhydrides]), elastomeric matrices, liposomes, microspheres,
oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and
groundnut), cocoa butter, waxes (e.g., suppository waxes),
paraffins, silicones, talc, silicylate, and the like.
[0210] Each carrier used in a pharmaceutical composition embodying
features of the present invention should be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not injurious to the animal. Carriers suitable for
a selected dosage form and intended route of administration are
well known in the art, and acceptable carriers for a chosen DDC,
dosage form and method of administration can be determined using
ordinary skill in the art. The pharmaceutical compositions
embodying features of the present invention may, optionally,
contain one or more additional ingredients and/or materials
commonly used in pharmaceutical compositions. These ingredients and
materials are well known in the art and include but are not limited
to (1) fillers or extenders, such as starches, lactose, sucrose,
glucose, mannitol, silicic acid or the like; (2) binders, such as
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
hydroxypropylmethyl cellulose, sucrose, acacia or the like; (3)
humectants, such as glycerol or the like; (4) disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, sodium starch glycolate,
cross-linked sodium carboxymethyl cellulose, sodium carbonate or
the like; (5) solution retarding agents, such as paraffin or the
like; (6) absorption accelerators, such as quaternary ammonium
compounds or the like; (7) wetting agents, such as cetyl alcohol,
glycerol monostearate or the like; (8) absorbents, such as kaolin,
bentonite clay or the like; (9) lubricants, such as talc, calcium
stearate, magnesium stearate, solid polyethylene glycols, sodium
lauryl sulfate or the like; (10) suspending agents, such as
ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and
sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar, tragacanth or the like; (11)
buffering agents; (12), excipients, such as lactose, milk sugars,
polyethylene glycols, animal and vegetable fats, oils, waxes,
paraffins, cocoa butter, starches, tragacanth, cellulose
derivatives, polyethylene glycol, silicones, bentonites, silicic
acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium
silicates, polyamide powder or the like; (13) inert diluents, such
as water, other solvents or the like; (14) preservatives; (15)
surface-active agents; (16) dispersing agents; (17) control-release
or absorption-delaying agents, such as hydroxypropylmethyl
cellulose, other polymer matrices, biodegradable polymers,
liposomes, microspheres; aluminum monostearate, gelatin, waxes or
the like; (18) opacifying agents; (19) adjuvants; (20) emulsifying
and suspending agents; (21), solubilizing agents and emulsifiers,
such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl
acetate, benzyl alcohol, benzyl benzoate, propylene glycol,
1,3-butylene glycol, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor and sesame oils), glycerol,
tetrahydrofuryl alcohol, polyethylene glycols, fatty acid esters of
sorbitan or the like; (22) propellants, such as
chlorofluorohydrocarbons or the like and volatile unsubstituted
hydrocarbons, such as butane, propane or the like; (23)
antioxidants; (24) agents which render the formulation isotonic
with the blood of the intended recipient, such as sugars, sodium
chloride or the like; (25) thickening agents; (26) coating
materials, such as lecithin or the like; and (27) sweetening,
flavoring, coloring, perfuming and preservative agents. Each such
ingredient or material should be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the animal. Ingredients and materials suitable for a
selected dosage form and intended route of administration are well
known in the art, and acceptable ingredients and materials for a
chosen DDC, dosage form and method of administration may be readily
determined by those of ordinary skill in the art.
[0211] Pharmaceutical formulations in accordance with the present
invention that are suitable for oral administration may be in the
form of capsules, cachets, pills, tablets, powders, granules, a
solution or a suspension in an aqueous or non-aqueous liquid, an
oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a
pastille, a bolus, an electuary or a paste. These formulations can
be prepared by methods well known in the art (e.g., by means of
conventional pan-coating, mixing, granulation or lyophilization
processes).
[0212] Solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules, and the like) may be
prepared by mixing the active ingredient(s) with one or more
pharmaceutically-acceptable carriers and, optionally, one or more
fillers, extenders, binders, humectants, disintegrating agents,
solution retarding agents, absorption accelerators, wetting agents,
absorbents, lubricants, and/or coloring agents. Solid compositions
of a similar type maybe employed as fillers in soft and hard-filled
gelatin capsules using a suitable excipient.
[0213] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using a suitable binder, lubricant, inert diluent,
preservative, disintegrant, surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine. The
tablets, and other solid dosage forms, such as dragees, capsules,
pills and granules, may optionally be scored or prepared with
coatings and shells, such as enteric coatings and other coatings
well known in the pharmaceutical-formulating art. They may also be
formulated so as to provide slow or controlled release of the
active ingredient therein. They may be sterilized by, for example,
filtration through a bacteria-retaining filter. These compositions
may also optionally contain opacifying agents and may be of a
composition such that they release the active ingredient only, or
preferentially, in a certain portion of the gastrointestinal tract,
optionally, in a delayed manner. The active ingredient can also be
in microencapsulated form.
[0214] Liquid dosage forms for oral administration include
pharmaceutically-acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. The liquid dosage forms may
contain suitable inert diluents commonly used in the art. Besides
inert diluents, the oral compositions may also include adjuvants,
such as wetting agents, emulsifying and suspending agents,
sweetening, flavoring, coloring, perfuming and preservative
agents.
[0215] Suspensions may contain suspending agents.
[0216] Formulations for rectal or vaginal administration may be
presented as a suppository, which may be prepared by mixing one or
more active ingredient(s) with one or more suitable nonirritating
carriers which are solid at room temperature, but liquid at body
temperature and, therefore, will melt in the rectum or vaginal
cavity and release the active compound. Formulations which are
suitable for vaginal administration also include pessaries,
tampons, creams, gels, pastes, foams or spray formulations
containing such pharmaceutically acceptable carriers as are known
in the art to be appropriate.
[0217] Dosage forms for topical or transdermal administration
include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches, drops and inhalants. The active compound may be
mixed under sterile conditions with a suitable
pharmaceutically-acceptable carrier. The ointments, pastes, creams,
and gels may contain excipients. Powders and sprays may contain
excipients and propellants. Pharmaceutical compositions suitable
for parenteral administrations may include one or more DDCs
embodying features of the present invention in combination with one
or more pharmaceutically-acceptable, sterile, isotonic, aqueous or
non-aqueous solutions, dispersions, suspensions or emulsions, or
sterile powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
suitable antioxidants, buffers, solutes which render the
formulation isotonic with the blood of the intended recipient, or
suspending or thickening agents. Proper fluidity may be maintained,
for example, by the use of coating materials, by the maintenance of
the required particle size in the case of dispersions, and by the
use of surfactants. These compositions may also contain suitable
adjuvants, such as wetting agents, emulsifying agents, and
dispersing agents. It may also be desirable to include isotonic
agents. In addition, prolonged absorption of the injectable
pharmaceutical form may be brought about by the inclusion of agents
which delay absorption.
[0218] In some cases, in order to prolong the effect of a drug, it
may be desirable to slow its absorption from subcutaneous or
intramuscular injection. This may be accomplished by the use of a
liquid suspension of crystalline or amorphous material having poor
water solubility. The rate of absorption of the drug then depends
upon its rate of dissolution which, in turn, may depend upon
crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug may be accomplished
by dissolving or suspending the drug in an oil vehicle. Injectable
depot forms maybe made by forming microencapsule matrices of the
active ingredient in biodegradable polymers. Depending on the ratio
of the active ingredient to polymer, and the nature of the
particular polymer employed, the rate of active ingredient release
can be controlled. Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue. The injectable materials can be
sterilized for example, by filtration through a bacterial-retaining
filter.
[0219] The formulations may be presented in unit-dose or multi-dose
sealed containers, for example, ampoules and vials, and may be
stored in a lyophilized condition requiring only the addition of
the sterile liquid carrier, for example water for injection,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of a type described above.
[0220] The present invention further provides methods of modulating
an integrin-mediated function of one or more cells. The methods
include contacting the cell(s) with an amount of a DDC embodying
features of the present invention that is effective to modulate the
integrin-mediated function. Methods of contacting cells in vivo are
the same as those described above for treating a disorder, disease
or condition. Methods of contacting cells in vitro with compounds
(e.g., placed in a solution, such as a cell culture medium,
containing the compound) are well known in the art. Suitable
conditions (time, temperature, concentrations, type of medium,
etc.) are known or can be determined empirically as is well known
in the art.
[0221] The present invention also provides methods of treating a
tissue by contacting the tissue with a DDC embodying features of
the present invention. Such treatment may improve the condition of
the tissue for subsequent use, as compared to tissue which is not
treated with a DDC. In particular, tissue which is to be
transplanted into a recipient may be treated with a DDC, preferably
before excision, or, if not, at the time of excision, and the
chances of the tissue being successfully transplanted may be
increased. The tissue to be treated may be any tissue. For example,
the tissue may be an organ tissue (such as tissue from a heart,
blood vessel, lung, liver, kidney, skin, cornea, or tissue from
part of an organ, such as a heart valve) or a non-organ tissue
(such as tissue from bone marrow, stern cells, or gametes). The
tissue is treated by contacting it one or more times with an
effective amount of a DDC embodying features of the present
invention. Methods of contacting tissues with agents are well known
in the art. For example, the contacting may be accomplished
conveniently by rinsing or perfusing the tissue with, and/or
submersing the tissue in, a solution of the DDC in a
physiologically acceptable diluent. Contacting may also include
perfusing the tissue or the donor of the tissue (e.g., a brain-dead
human) with a solution of the DDC in a physiologically acceptable
diluent prior to excision of the tissue. Physiologically-acceptable
diluents are those that are compatible with, and not harmful to,
the DDC and the tissue. Such diluents are well known and include
saline and other solutions and fluids.
[0222] Effective amounts of a DDC embodying features of the present
invention may be determined empirically, and making such
determinations lies well within the skill of the ordinary artisan.
It is well understood within the art that the amount may vary as a
result of one or more factors, including the type and size of the
tissue, the intended use of the tissue, the length of storage of
the tissue before use, the identity of any other agents being used,
the number of treatments, and like factors well known in the
art.
[0223] A DDC agent embodying features of the present invention may
be used in conjunction with other agents to treat tissue. For
example, the tissue may also be treated with preservation agents
(i.e., agents which inhibit deterioration of the condition of the
tissue), antibiotics, antifungal drugs, antiviral drugs, and
inflammatory drugs, or other treatments (e.g., lung surfactants in
the case of lung tissue). After being contacted with the DDC, the
tissue may be used immediately or may be stored until needed.
Methods of storing tissue are well known in the art. The tissue may
be stored in contact with the DDC. Tissues are preferably stored at
low temperatures, typically from 4-18.degree. C., and non-organ
tissues may typically be frozen. The time of storage will vary
depending on the type of tissue, the storage environment (including
the temperature of storage), and the intended use. Such times may
be determined empirically, and making such determinations lies well
within the skill of the ordinary artisan. Regardless of the length
and conditions of storage, timely treatment with a DDC embodying
features of the present invention may mitigate the effects of
harvest and/or storage, and treated tissues may be in better
condition than tissues not treated with a DDC.
[0224] Tissues treated with DDCs embodying features of the present
invention may be used for a variety of purposes. For example, they
may be transplanted into recipients. They may also be used for
research purposes, such as for studying the function of the
tissue.
[0225] It is understood that DDCs may induce a rest state in
tissue, which lasts until the DDCs are removed. In addition,
treatment of a tissue with a DDC embodying features of the present
invention may improve the condition of the tissue by reducing the
negative effects and consequences of harvesting and storing tissue.
For example, DDCs may inhibit (prevent or reduce) the adhesion and
aggregation of cells which would otherwise cause injury to a tissue
(see description above). Thus, treatment of a tissue with a DDC
embodying features of the present invention may prevent or reduce
damage to the tissue.
[0226] In particular, ischemia (anemia due to constriction or
obstruction of a blood vessel) occurs upon harvesting a tissue or
an organ. Both the injury due to ischemia and that due to
reperfusion after ischemia (which generally occurs upon resuming
blood flow in an organ, such as when transplanting a tissue or an
organ into a recipient), may be inhibited by treatment of the
tissue or organ with a DDC embodying features of the present
invention. To achieve maximum inhibition of ischemic injury and
ischemic reperfusion injury, the tissue or organ may be contacted
with the DDC before, during or after harvesting of or interruption
of normal blood supply to the tissue or organ, to mitigate the
rapid onset of injury and other changes associated with ischemic
injury. Similarly, the tissue or organ may be contacted with the
DDC before, during or after transplantation. Such treatment may
have beneficial effects even for organs that are to be used
immediately, such as in the case of many transplants. Preferably
the contacting takes place by perfusion of the organ with a
solution comprising the DDC, although rinsing and/or submersion of
the tissue or organ in a medium containing the DDC may also be
beneficial.
[0227] For tissues or organs that are stored (even for a short
time), benefits may be obtained by contacting the tissue or organ
with a DDC embodying features of the present invention immediately
prior to use. This treatment may serve, among other purposes, to
eliminate the effects of any cytokines that may have been produced,
as well as to prevent adhesion and aggregation of cells which would
otherwise cause tissue or organ injury. The treatment of a tissue
or organ after storage may be the first treatment of the tissue or
organ with a DDC, or may be the second treatment of the tissue or
organ which was fast treated before or immediately after harvest.
Again, the organ may be preferably treated by perfusion with a
solution containing the DDC, although rinsing and/or submersion of
the tissue or organ in a medium containing the DDC may also be
beneficial.
[0228] As described above, DDCs embodying features of the present
invention may suppress undesired immune responses. Thus, treatment
of a tissue with a DDC prior to transplantation may act as an
initial treatment for the prevention of graft rejection and/or
graft versus host disease (GVHD) in transplant recipients. Of
course, the recipient may receive additional amounts of a DDC in
accordance with the present invention, either in single dose
treatments or in multiple dose treatments, to prevent graft
rejection and/or GVHD as described above. The amount administered
to the recipient should also be chosen so that inhibition of injury
due to ischemia and ischemia-reperfusion is continued.
[0229] Furthermore, as described above, DDCs embodying features of
the present invention, administered as described above, may inhibit
the transmission of pathogenic and/or viral infections from a
tissue to a recipient of transplanted tissue and vice versa. This
includes-all of the viral infections described above.
[0230] The invention also provides a kit for treating tissues. As
used herein, the terms "kit" and "reagent kit" refer to an assembly
of materials that are used in performing a method embodying
features of the present invention. The reagents may be provided in
packaged combination in the same or in separate containers,
depending on their cross-reactivities and stabilities, and in
liquid or in lyophilized form. The amounts and proportions of
reagents provided in the kit may be selected so as to provide
optimum results for a particular application. A reagent kit
embodying features of the present invention contains at least one
DDC embodying features of the present invention.
[0231] Reagents included in kits embodying features of the present
invention may be supplied in all manner of containers such that the
activities of the different components are substantially preserved,
while the components themselves are not substantially adsorbed or
altered by the materials of the container. Suitable containers
include but are not limited to ampoules, bottles, test tubes,
vials, flasks, syringes, bags and envelopes (e.g., foil-lined), and
the like. The containers may be comprised of any suitable material
including but not limited to glass, organic polymers (e.g.,
polycarbonate, polystyrene, polyethylene, etc.), ceramic, metal
(e.g., aluminum), metal alloys (e.g., steel), cork, and the like.
In addition, the containers may contain one or more sterile access
ports (e.g., for access via a needle), such as may be provided by a
septum. Preferred materials for septa include rubber and polymers
including but not limited to, for example, polytetrafluoroethylene
of the type sold under the trade name TEFLON by DuPont (Wilmington,
Del.). In addition, the containers may contain two or more
compartments separated by partitions or membranes that can be
removed to allow mixing of the components.
[0232] Kits embodying features of the present invention may also be
supplied with other items known in the art and/or which may be
desirable from a commercial and user standpoint, such as
instructions for treating a tissue, a container for the tissue,
diluents, preservation agents, antibiotics, antifungal drugs,
antiviral drugs, anti-inflammatory drugs, surfactants, buffers,
empty syringes, tubing, gauze, pads, disinfectant solution, etc.
Instructional materials provided with kits embodying features of
the present invention may be printed (e.g., on paper) and/or
supplied in an electronic-readable medium (e.g., floppy disc,
CD-ROM, DVD-ROM, zip disc, videotape, audio tape, etc.).
Alternatively, instructions may be provided by directing a user to
an Internet web site (e.g., specified by the manufacturer or
distributor of the kit) and/or via electronic mail.
[0233] It should be understood that whereas some integrin-mediated
functions may be modulated by DDCs of formulae (10)-(12), (35), and
(40), there may be certain integrin-mediated functions, the
modulation of which is achieved by other CIMs and/or DDCs,
including but not limited to one or more enantiomers of ajoene,
derivatives thereof including the subgroup of (2Z)-(-)-, (2Z)-(+)-,
(2E)-(-)-, and (2E)-(+)-DDCs of formula (9), compounds of formulae
(2) and/or (2a), and the like. Accordingly, kits embodying features
of the present invention may contain all manner of integrin
modulators including trithia and/or dithia oxide structures and
chiral structures, such as those described above.
[0234] The following representative procedures and Examples are
provided solely by way of illustration, and are not intended to
limit the scope of the appended claims or their equivalents.
EXAMPLES
Example 1
Methods of Ajoene Separation into Four Enantiomers
[0235] A 3:1 mixture of racemic (Z)-(.+-.)- and racemic
(E)-(.+-.)-ajoenes (unseparated ajoene) was dissolved in
heptane/ethanol/diethylamine (90:10:0.1) and separated by HPLC on a
4.6.times.250 mm Chiralpak AS column (Chiral Technologies, Exton,
Pa.) using a mobile phase of heptane/ethanol/diethylamine 90:10:0.1
(flow rate, 1 mL/min; temperature, 25.degree. C.). The detector
recorded two parameters: the absorption of the eluate at 254 nm and
the rotation of the plane of polarization of polarized light
passing through the eluate; in the latter case, upward and downward
peaks corresponded, respectively, to dextrorotatory (+) and
levorotatory (-) enantiomers. The resulting chromatogram contained
eight, peaks superimposed in such a way that four pairs were
clearly seen. In each pair, the two coinciding peaks corresponded
to a particular enantiomer of ajoene--that is, (Z)-(+), (Z)-(-),
(E)-(+), and (E)-(-). The (Z)-(+), (E)-(+), (E)-(-), and (Z)-(-)
enantiomers of ajoene were eluted with retention times of 21.4,
23.8, 26.2, and 33.1 minutes, respectively.
[0236] In another experiment, the separation was achieved in the
same system, the only difference being in the composition of the
mobile phase (hexane/ethanol 90:10). Again, the (Z)-(+), (E)-(+),
(E)-(-), and (Z)-(-) enantiomers were clearly separated (the
respective elution times were 20.4, 23.1, 24.6, and 31.2 minutes).
The peaks were collected into tared polypropylene tubes and stored
on ice for 48 hours, after which analytical runs were performed
with each fraction. The results of the analysis demonstrated that
very good separation of the isomers had been achieved (>98%) and
that the isomers did not undergo change in optical or geometric
configuration under the conditions of the storage. Thereafter, the
fractions were dried down by rotary evaporation (6 to 10 min at
35.degree. C.) and re-analyzed under the same conditions. The peaks
present in the fractions showed their original retention times and
no other peaks were present. Thus, the drying down process did not
cause change in optical or geometric configuration.
[0237] In yet another experiment, HPLC separation of the
enantiomers involved a different column (10.times.250 mm Chiralpak
AD). The conditions used were as follows: mobile
phase=hexane/ethanol 90:10; flow rate=6 mL/min;
temperature=ambient; detector wavelength=254 nm. Typically, 10 mg
of the 3:1 mixture of ajoenes was loaded per run and fractions were
collected manually. The fractions were dried down by rotary
evaporation at 37.degree. C., resulting in light oil. The oil was
taken up in anhydrous ether and dried down by rotary evaporation.
The resulting light oils were stored at -80.degree. C. in sealed
containers. Analytical runs on the same column demonstrated that
(Z)-(+), (E)-(+), (E)-(-), and (Z)-(-) enantiomers of ajoene had
retention times of 53 min, 58 min, 62 min, and 75 min,
respectively.
Example 2
Inhibition of VLA-4-Mediated Cell Adhesion: a Method of Assessment
of Integrin-Mediated Function Modulating Activity of the Four
Enantiomers of Ajoene
[0238] Modulating activity of integrin-mediated functions by the
enantiomers was compared in a well-defined system of inhibition of
VLA-4-mediated adhesion of enzyme-labeled PMI cells (NIH AIDS
Repository, Rockville, Md.) to VCAM-1-coated artificial substrata.
In this experiment, enzyme-linked immunosorbent assay (ELISA)
plates were coated with rabbit anti-human IgG (Fc-specific).
Aliquots of 100 .mu.L of supernatant from VCAM/IgG-secreting COST
cells were added to each well, the plates incubated at 37.degree.
C. for 1 hour, and the wells washed with phosphate-buffered saline
(PBS). Thereafter, 50 .mu.L aliquots of the enantiomers (various
dilutions in RPMI 1640 medium) were introduced to each well,
followed by addition of 50 .mu.L PMI cells (4.times.10.sup.6 per
mL) labeled with horseradish peroxidase (HRP) by pinocytosis.
[0239] The plates were incubated at room temperature for 10 min,
centrifuged (1000 rpm, 1 min) and incubated once again at
37.degree. C. for an additional 10 min. Cells that failed to form
VLA-4-dependent adhesive contacts with the substratum were washed
off with PBS in two turns. Adherent cells were lyzed by adding to
each well a buffered solution of the substrate, supplemented with
1% Triton X-100. The reaction was stopped with 0.5 M
H.sub.2SO.sub.4, and the optical density of the wells was read at
450 nm. The value of this parameter, characterizing the activity of
the enzyme and the PCT/US2005/007638 number of the adherent cells,
is inversely proportional to the modulating activity of the
compound. The (Z)-(-) enantiomer of ajoene consistently exhibited a
4-fold higher adhesion-inhibiting activity than any other
enantiomer of the original 3:1 mixture of racemic Z- and racemic
E-ajoenes.
Example 3
Differential Inhibition by Ajoene Enantiomers of Integrin-Mediated
Fusion Leading to Syncytium Formation in HIV-Infected Cells
[0240] The purified enantiomers were taken up in DMSO at a
concentration of 10 mg/mL. Dilutions were made in RPMI 1640 medium
containing 10% fetal calf serum and 10 mM HEPES (cRPMI). H9 cells
(ATCC, Rockville, Md.) infected with HIV-1.sub.RF (NIH AIDS
Repository, Rockville, Md.) and uninfected H9 cells were washed
with cRPMI and resuspended in cRPMI at a density of
4.times.10.sup.6 per mL. Uninfected cells (50 .mu.L) were mixed
with serial dilutions of the compounds (100 .mu.L) and incubated at
37.degree. C. for 30 min before adding and mixing 50 .mu.L of
infected H9 cells. The plates were incubated at 37.degree. C. for 6
to 15 hours before scoring syncytium formation.
[0241] The results are presented in FIG. 1. For all the curves, the
effect of ajoene enantiomers and enantiomer mixtures on the fusion
of cultured, intact H9 cells with HIV-1.sub.RF-infected H9 cells is
disclosed. The vertical graph axis expresses the maximum amount of
syncytia formed in the absence of the compounds (100 percent),
while the points on the curves represent percentages of such an
amount of syncytia formed in the presence of varying concentrations
of the compounds and compound mixtures (micromoles per liter). The
coincidence of curves 1 through 3 and the leftward shift of curve 4
demonstrate that the (Z)-(-)-enantiomer is at least four times more
active than its (Z)-(+) counterpart, racemic (E)-ajoene, or the
unseparated 3:1 mixture of racemic (Z)- and racemic (E)-ajoenes
(the respective values of IC.sub.100 are 12.5 and 50 micromoles per
liter).
[0242] A similar result was obtained in another system, where
syncytium formation was induced by mixing and incubating together
for 13 hours 50 .mu.L MT2 cells (NIH AIDS Repository) and 50 .mu.L
U937 cells (ATCC) infected with HIV-1 RT (both cell populations had
the density of 2.times.10.sup.6 per mL). Taken together, Examples 2
and 3 demonstrate that
(Z)-(-)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide is a
stereoselective, chiral integrin modulator, the activity of which
is significantly higher than that of the parent racemates (E-,
Z-ajoenes, and various mixtures thereof).
Example 4
Bioavailability of DDCS and the Active Moiety of Chiral Ajoene
[0243] As indicated in the foregoing Examples, the modulating
activity of ajoene differs depending on the chiral arrangement of
the sulfoxide and the geometry of the C6 double bond. Indeed, as
described below, the metabolically unrelated
CH.sub.2.dbd.CH--CH.sub.2--S-- (allylthio) group of the molecule,
shared by all four enantiomers, is routinely severed in biological
media, with the chiral integrin-mediated function modulating moiety
remaining bioavailable in relation to the activities described
herein, including in the foregoing Examples.
[0244] Thus, the allylthio fragment of the ajoene molecule may be
replaced, in the presence of a sulfhydryl compound (HS--R), by a
suitable R with the formation of DDCs of the formula:
##STR00033##
[0245] As illustrated below, an example compound of this formula is
ajocysteine (a), one molecule of which is formed via the
interaction of one molecule of ajoene (b) with two molecules of
cysteine (c). In this reaction, the allylthio moiety of ajoene is
converted to one molecule of S-allylmercaptocysteine (d) (see:
"Garlic: The Science and Therapeutic Application of Allium Sativum
L. and Related Species," Lawson, L. D. et al., Eds., 1997, p.
64).
##STR00034##
[0246] The scheme shown below illustrates one specific
metabolically related setting, in which ajoene (a) is converted
into DDCs when exposed to blood components; the DDC that may be
formed in this reaction is (e). Allylmercaptan (c), which is
derived from the allylthio fragment of ajoene (Ibidem, pp.
213-214), is unstable and undergoes enzymatic transformation to
allyl methyl sulfide (d); formula (b) designates an unidentified
reactant in the blood. Allyl methyl sulfide is a volatile
metabolite discharged with breath (R. T. Rosen et al.,
"Determination of allicin, S-allylcysteine and volatile metabolites
of garlic in breath, plasma or simulated gastric fluids," J. Nutr.,
2001, 131, [3S] 968S; and "Garlic: The Science and Therapeutic
Application of Allium Sativum L. and Related Species" cited above,
p. 66).
##STR00035##
[0247] All four possible enantiomers of ajoene have a capacity to
form the corresponding (2E)-(+)-, (2E)-(-)-, (2Z)-(+)-, and
(2Z)-(-)-DDCs, both in vitro and in vivo.
[0248] Generally, any compound containing the core trithia oxide
substructure of formula (1) is believed to be capable of forming
derivatives of the following structure:
##STR00036##
[0249] As indicated above, these compounds include all isomeric
possibilities set forth herein, including enantiomeric,
constituting a distinct subgroup of DDCs in accordance with the
present invention.
Example 5
Synthesis of DDCS of General Formula (10)
[0250] A representative synthetic approach to compounds of general
formula (10) in which m=0 and n=1 is illustrated in Scheme 1
below:
##STR00037##
[0251] In the representative Scheme 1 shown above, a compound
X.sup.1H (13) is reacted with prop-2-ene-1-sulfinyl chloride (15)
to form a sulfoxide-containing compound (16). The sulfinyl chloride
(15) may be prepared from commercially available 2-propene-1-thiol,
available from Aldrich (Milwaukee, Wis.) and other suppliers (e.g.,
Alfa Aesar, Fluka, Lancaster, Acros Organics, Pfaltz & Bauer),
by oxidation followed by treatment with a chlorinating agent (e.g.,
thionyl chloride). Other useful starting materials analogous to
sulfinyl chloride (15) include 1-alkenesulfinyl chlorides, such as
those reported in the literature (e.g., Journal of Organic
Chemistry 1998, 63, 7825-7832).
[0252] Depending on the structure of the X.sup.1 group to be
contained in the final product, compound (13) may either be reacted
with sulfinyl chloride (15) directly or by preparing an
intermediate compound (14), which may correspond to a salt of
compound (13). For example, if the X.sup.1 group of compound (13)
is to be attached to the sulfoxide sulfur atom through a
nucleophilic atom (e.g., oxygen, nitrogen, etc.), it may be
desirable to react compound (13) with compound (15) directly,
optionally in the presence of a catalyst (e.g., pyridine).
Moreover, in certain instances, it may be desirable to remove an
acidic proton on the X.sup.1 group of compound (13) (e.g., by
deprotonation with a base) to form a salt (14), which is then
reacted with sulfinyl chloride (15). The M group in compound (14)
may correspond to a wide variety of metals, including but not
limited to Na, Mg, Li, Cu, and the like.
[0253] Sulfoxide-containing compound (16) is halogenated, for
example with N-chlorosuccinimide (NCS), to provide chloro compound
(17). Chloro compound (17) is converted to thiol (18) by reaction
with, for example, excess NaSH, thiourea/water or the like. The
thiol (18) is then reacted with a compound (19) or the like to
introduce X.sup.2 and provide the final DDC product (20), which
corresponds to general formula (10) in which m=0 and n=1. Depending
on the structure of the X.sup.2 to be contained in the final
product, the conversion of thiol (18) to DDC (20) may proceed
simply using substitution-type chemistry (e.g., when the X.sup.2
portion of compound (19) contains a suitable leaving group, such as
mesylate, tosylate or the like).
[0254] A representative synthetic approach to compounds of general
formula (10) in which m=1 and n=0 is illustrated in Scheme 2
below:
##STR00038##
[0255] In the representative Scheme 2 shown above, a compound (20)
containing a leaving group (e.g., -OMs) is reacted with
commercially available 2-propene-1-thiol (21) to form compound
(22). The double bond of compound (22) is brominated and then
treated with base to form vinyl bromide (23) with the loss of HBr.
Vinyl bromide (23) is converted to a Grignard reagent by treatment
with magnesium, and the corresponding Grignard reagent is quenched
with sulfur to form vinyl thiol (24). Vinyl thiol (24) is reacted
with a compound such as X.sup.2OMs (25) or the like to form a
disulfide compound (26). Selective oxidation of compound (26) forms
final product (27), which corresponds to general formula (10) in
which m=1 and n=0. The selective oxidation of one of the sulfur
atoms in the presence of the other may be aided by steric
accessibility of the sulfur to be oxidized. However, mixtures of
oxidized products may be separated to obtain the desired oxidized
product, as will be appreciated by those of ordinary skill in the
art.
[0256] This above-described representative Scheme 2 may be modified
in numerous ways, as will be appreciated by one of ordinary skill
in the art. For example, in the synthesis of compound (27) shown
above, the Grignard reagent prepared from vinyl bromide (23) may be
reacted with a compound having a structure X.sup.2S--SX.sup.2 to
provide compound (26) directly.
[0257] An alternative representative synthetic approach to
compounds of general formula (10) in which m=1 and n=0 is shown in
Scheme 3 below:
##STR00039##
[0258] In the representative Scheme 3 shown above, propargyl thiol
(28) is converted to propargyl sulfinyl chloride (29) by oxidation
followed by treatment with a chlorinating agent (e.g., thionyl
chloride). Propargyl thiol (28) may be prepared as reported in the
literature (e.g., Synthesis 1997, 518-520). Propargyl sulfinyl
chloride (29) is alkylated with a compound (13) to form
sulfoxide-containing alkyne (30). Hydroboration of compound (30)
(e.g., with catecholborane) and trapping with bromine gives vinyl
bromide (31). Vinyl bromide (31) is converted to a Grignard reagent
by treatment with magnesium, and the corresponding Grignard reagent
is quenched with sulfur to form vinyl thiol (32). Dilute reaction
concentrations may be employed to minimize or prevent potential
undesired side reactions between Grignard moieties and sulfoxide
moieties. Vinyl thiol (32) is then reacted with compound (25) to
form final product (27).
[0259] The above-described representative Scheme 3 may be modified
in numerous ways, as will be appreciated by one of ordinary skill
in the art. For example, the sulfoxide moiety in compound 31 may be
protected prior to formation of the Grignard reagent in order to
prevent potential undesired reactions at the sulfoxide group.
[0260] It is to be understood that the above-described syntheses
are merely representative approaches that may be modified in
numerous ways, as will be appreciated by those of ordinary skill in
the art. All manner of chemical transformations and reagents known
in the art are contemplated for use in accordance with the
presently preferred embodiments--including but not limited to those
described in treatises such as Comprehensive Organic
Transformations, 2.sup.nd Edition by Richard C. Larock (Wiley-VCH,
New York, 1999), Advanced Organic Chemistry Part B: Reactions and
Synthesis by Francis A. Carey and Richard J. Sundberg (Kluwer
Academic/Plenum Publishers, 2001), Some Modern Methods of Organic
Synthesis, 3.sup.rd Edition by W. Carruthers (Cambridge, 1987),
Protective Groups in Organic Synthesis, 3.sup.rd Edition by
Theodora W. Greene and Peter G. M. Wuts (John Wiley & Sons,
Inc., 1999), and March's Advanced Organic Chemistry, 5.sup.th
Edition by Michael B. Smith and Jerry March (John Wiley & Sons,
Inc., 2001), and references cited therein. The entire contents of
all of the above-identified treatises are incorporated herein by
reference, except that in the event of any inconsistent disclosure
or definition from the present application, the disclosure or
definition herein shall be deemed to prevail.
[0261] In addition, the general synthetic approaches outlined above
can be readily modified for use in the preparation of compounds in
which the values of m and/or n are integers other than 0 and 1 by
the choice of different starting materials analogous to sulfinyl
chloride (15), 2-propene-1-thiol (21), and propargyl sulfinyl
chloride (29).
Example 6
DDC Attachment to Polymeric Carriers: an Approach to Targeted
Delivery
[0262] An additional group of DDCs embodying features of the
present invention may be synthesized, the compounds of which share
two common characteristics: (a) the polymeric nature of X.sup.2 and
(b) multiplicity of the active moieties attached to a single
X.sup.2. These compounds have a general formula:
##STR00040##
[0263] For such compounds, X.sup.2 will serve as an appropriate
carrier delivering the active moiety (a DDC moiety or a derivative
thereof) to a designated site within an organism. In this context,
appropriate carriers include but are not limited to natural or
synthetic polysaccharides, proteins, polypeptides or peptides,
polymers of other types not encountered in nature (e.g., polymers
of fluorinated hydrocarbons), and the like. In addition, a solid
phase binding group (e.g. biotin) may be used to couple a DDC
functionality or a derivative thereof to a carrier (e.g., avidin,
streptavidin).
[0264] For example, a compound of formula (11), wherein X.sup.2 is
a polysaccharide carrier, may be a polymer of furanose and/or
pyranose units (optionally modified with amino or sulfhydryl groups
and/or residues of acetic or sulfuric acids) carrying N-, C-, or
S-linked DDC functions or their derivatives.
[0265] Furthermore, compounds of formula (11), wherein X.sup.2 is a
polypeptide, may include synthetic structures, the cysteine
residues of which carry S-linked DDC functions or their
derivatives.
[0266] The choice of a carrier creates a possibility to either
facilitate the release of the active moiety or, alternatively, make
the bond between it and the carrier stable. Moreover, compounds of
formula (11) provide a convenient means of targeted delivery of DDC
moieties. For example, a polysaccharide DDC may be conjugated with
proteins (to create glycoproteins carrying DDCs), or engineered to
contain specific oligosaccharide units recognized by cell surface
lectins. Likewise, a polypeptide alkylated with DDC moieties may be
conjugated with antibodies recognizing specific cell/tissue
markers. By using this approach, DDCs may be targeted to
intracellular compartments.
[0267] Compounds of formula (11) may be prepared using a variety of
synthetic approaches, including but not limited to strategies
analogous to those outlined in Schemes 1-3 above. One
representative synthetic approach to compounds of formula (11) is
shown in Scheme 4 below:
##STR00041##
[0268] In the representative Scheme 4 shown above, a plurality (w)
of DDC thiols (33) are reacted with a carrier (34) that contains a
plurality (z) of leaving groups (LG). Preferably, the carrier (34)
contains at least as many leaving groups as the number of DDC
fragments to be attached thereto (i.e., z.gtoreq.w). In this
representative Scheme 4, attachment of DDC fragments to the carrier
is achieved by substitution-type chemistry analogous to the
preparations of compounds (20), (26), and (27) shown above in
Schemes 1, 2, and 3, respectively. It should be noted that the
individual DDC thiols (33) to be attached to the carrier (34) need
not be identical and that the plurality of compounds (33) may
include compounds containing X.sup.1 groups that are the same or
different. Similarly, the m and/or n values of the plurality of
compounds (33) may be the same or different. Furthermore, the
individual DDC thiols (33) may include mixtures of stereoisomers or
individual isomers, such as the (E,R), (E,S), (Z,R), and (Z,S)
isomers.
Example 7
Polymeric DDCs
[0269] An additional group of DDCs embodying features of the
present invention may be synthesized in which a plurality of DDC
moieties, which may be the same or different, are incorporated into
a polymeric structure. These compounds have a general formula:
##STR00042##
[0270] The variables m, n, X.sup.1, and X.sup.2 may be as defined
above for the DDCs embodying features of the present invention. The
groups X.sup.1 and X.sup.2 may be the same or different and include
at least one functional group, preferably at a terminal position,
which is capable of participating in a polymerization-type reaction
(e.g., chain-growth, step-growth, etc.) either with a functional
group within the same monomeric unit or with a functional group in
a different monomeric unit. The variable w is an integer from 2 to
1000.
[0271] One representative synthetic approach to compounds of
general formula (35) is shown in Scheme 5 below:
##STR00043##
[0272] In the representative Scheme 5 shown above, a DDC compound
(36) is reacted with an .alpha.,.omega.-diol (37) to form a
polymeric structure (38). Each of the X.sup.1 and X.sup.2 portions
of compound (36) contains a carboxylic acid, preferably a
terminally located one, which may react with one of the hydroxyl
functions of the .alpha.,.omega.-diol (37), thereby forming an
ester linkage. The distance between DDC moieties in the polymeric
backbone may be controlled by varying the length of the alkylene
portion of the .alpha.,.omega.-diol (37). Preferably, the variable
i in .alpha.,.omega.-diol (37) is between 1 and 30. In addition, it
is presently preferred that the X.sup.1 and X.sup.2 moieties be
sufficiently long to minimize or prevent any adverse interaction
with the adjacent carbonyl groups that could otherwise reduce the
efficacy of the DDC units in the polymeric chain. In addition, it
is presently preferred that the X.sup.1 portion of compound (36) is
alkenyl and that at least one double bond in the alkenyl X.sup.1
fragment is allylic to the sulfoxide group in order to resemble the
structure of the terminal double bond allylic to the sulfoxide
group in ajoene. Thus, a presently preferred structure for compound
(36) has a formula:
##STR00044##
wherein X.sup.1a may be as defined above for the X.sup.1 portion of
DDCs embodying features of the present invention.
[0273] It should be noted that while the polymerization chemistry
illustrated in representative Scheme 5 above is based on a
step-growth polymerization with formation of polyester linkages,
all manner of polymerizations known in the art (e.g., formation of
polyamides, polycarbonates, polyurethanes, epoxy resins,
polymerizations of alkenes and alkynes, free radical
polymerizations, anionic polymerizations, cationic polymerizations,
and the like) may be adapted to and are contemplated for use in
accordance with the present invention. Representative
polymerizations useful in accordance with the present invention
include but are not limited to those described in the treatise
entitled Principles of Polymerization, 4.sup.th Ed. By George Odian
(John Wiley & Sons, Inc., New York, 2004), the entire contents
of which are incorporated herein by reference, except that in the
event of any inconsistent disclosure or definition from the present
application, the disclosure or definition herein shall be deemed to
prevail.
[0274] An additional group of polymeric DDCs embodying features of
the present invention has a general formula:
##STR00045##
[0275] The plurality (w) of DDC moieties in compound (40) may be
the same or different. The variables m, n, and X.sup.1, and w may
be as defined above. In the compounds of formula (40), the sulfur
atom of one DDC moiety is directly attached to the X.sup.1 group of
another DDC moiety. The group X.sup.1 includes at least one
functional group, preferably at a terminal position, which is
capable of participating in a polymerization-type reaction (e.g.,
chain-growth, step-growth, etc.) with a sulfur atom.
[0276] One representative synthetic approach to compounds of
general formula (40) is shown in Scheme 6 below:
##STR00046##
[0277] As shown in the representative Scheme 6 above, a dimeric DDC
(42) may be prepared by the intermolecular coupling of two
molecules of thiol (41). The X.sup.1 portion of thiol (41) contains
a suitable leaving group LG (e.g., mesylate, tosylate or the like),
which may react with the thiol end of a second molecule, or the
salt thereof, by substitution-type chemistry. The dimeric DDC (42)
may then be further reacted with additional thiol (41) or a salt
thereof to introduce a third DDC subunit, thereby increasing the
length of the oligomeric chain. Undesired intramolecular
cyclization of dimer (42) or higher oligomers may be minimized or
prevented by the reaction conditions used (e.g., concentrations of
reactants). In addition, the geometry of the central double bond in
thiol (41) and/or the structure of the X.sup.1 moiety may likewise
decrease the possibility of competitive intramolecular cyclizations
(e.g., an E configuration for the central double bond of thiol (41)
and dimer (42) will hold the reactive centers apart, thereby
reducing the tendency towards intramolecular cyclization).
[0278] It should be noted that while the coupling chemistry
illustrated in representative Scheme 6 above is based on a
step-wise increase of the oligomeric chain using substitution-type
couplings, additional methodologies, such as the addition of thiols
to alkenes or alkynes (e.g., by electrophilic, nucleophilic or
free-radical mechanisms), may also be employed and are likewise
contemplated for use in accordance with the present invention. In
these synthetic approaches, the X.sup.1 portion of thiol (41) may
contain a double or triple bond instead of leaving group LG.
Example 8
DDC Attachment to Particulate Material
[0279] In certain cases, targeted delivery is most efficient when
particulate material (e.g., microspheres, beads, liposomes,
micelles, etc.) to which the active agents are attached is used as
a carrier. Polymeric DDCs of formula (11) may be readily
immobilized on surfaces of beads, using techniques developed for
(poly)peptides, (poly)saccharides, and (glyco)proteins. Obtaining
stable preparations of liposomes carrying DDCs may involve their
conversion into lipids capable of forming liposomes (i.e.,
phospholipids, sphingolipids, glycolipids, or sterols).
[0280] For example, if X.sup.1 in formula 10 is allyl, X.sup.2
should be selected from substituted phosphoglyceryls,
furanose/pyranose units, and polycyclic aryls, in order to obtain,
respectively, a phospholipid/sphingolipid, a glycolipid, or a
sterol modified with a DDC function (e.g., via S-alkylation,
N-alkylation, O-alkylation, or O-acylation). Incorporation of such
modified lipids into liposomes or micelles gives a particulate form
of DDCs having the formula:
##STR00047##
Example 9
Dimeric DDCs Linked Through a Disulfide Bond
[0281] An additional group of DDCs embodying features of the
present invention may be synthesized in which two DDC moieties,
which may be the same or different, are incorporated into a dimeric
structure linked through a disulfide bond. These compounds have a
general formula (43):
##STR00048##
[0282] The variables X.sup.1 and X.sup.2 may be the same or
different and may be as defined above for the DDCs embodying
features of the present invention. The variables m, n, o, and p may
each be independently selected from the group of integers from 0 to
30. In a presently preferred embodiment, X.sup.1=allyl, m=1, n=0,
X.sup.2=allyl, p=1, and o=0, such that the compound (43) represents
a dimeric structure containing two DODOyl fragments corresponding
to the active moieties of ajoene.
[0283] It is to be understood that compounds of general formula
(43) include all of the possible optical and geometric isomers.
Accordingly, cleavage of the disulfide bond of compound (43) may
result in the formation of several different active moieties having
different chiral and/or geometric configurations, depending on the
chiral and/or geometric purity of the starting compound. As used
herein, isomers of compound (43) are identified sequentially from
left to right by (1) the configuration of the central sulfoxide in
the left half of the molecule, (2) the configuration of the central
double bond in the left half of the molecule, (3) the configuration
of the central double bond in the right half of the molecule, and
(4) the configuration of the central sulfoxide in the right half of
the molecule. For example, a specific isomer of compound (43) will
be designated as (R or S, E or Z, E or Z, R or S).
[0284] Compounds of general formula (43) may be readily prepared
from the corresponding monomeric thiols by oxidation (e.g., with
H.sub.2O.sub.2, CuSO.sub.4, Me.sub.2SO--I.sub.2 or the like) or by
alternative methodologies including but not limited to the reaction
of the corresponding monomeric alkyl halides with disulfide
anions.
[0285] DDCs of general formula (43) may have particular therapeutic
utility inasmuch as one molecule of general formula (43) provides a
"double dose" of DDC moieties. The individual DDC units may be
readily liberated by cleavage of the disulfide bond.
Example 10
Dimeric DDCS Linked Through an Intermediary Moiety
[0286] An additional group of DDCs embodying features of the
present invention, which are related to compounds of formula (43)
above, may be synthesized in which two DDC moieties, which may be
the same or different, are incorporated into a dimeric structure
linked through an intermediary moiety X.sup.3. These compounds have
a general formula (44):
##STR00049##
[0287] The variables X.sup.1 and X.sup.2 may be the same or
different and may be as defined above for the DDCs embodying
features of the present invention. The variables m, n, o, and p may
each be independently selected from the group of integers from 0 to
30. The variable X.sup.3 is a linking moiety capable of forming
bonds (e.g., covalent and/or ionic) with sulfur atoms of the two
DDC moieties. By way of example, X.sup.3 may be a divalent moiety
such as an alkylene group, for example --(CH.sub.2).sub.q--, where
q is an integer from 1 to 30. Such compounds may be readily
prepared from the corresponding thiols by substitution-type
chemistry, as described above. Alternatively, X.sup.3 may be a
positively charged cationic species, such as a metal atom,
preferably with a charge of at least +2, to counterbalance the
negative charges of the two thiolate anions (i.e., S.sup.-) of the
DDC portions of the molecule. Such compounds may be prepared by
deprotonating the thiol hydrogens in the corresponding thiols to
form thiolate anions, and by forming a salt with a suitable cation
(e.g., Zn.sup.2+, Fe.sup.2+, Cu.sup.2+, Co.sup.2+, etc.). Of
course, cationic species having charges other than +2 may also be
used, including but not limited to cations with charges of +3 and
+4. With such higher charge cations, three or more DDC units may be
associated with the salts of general formula (44).
[0288] In a presently preferred embodiment, X.sup.1=allyl, m=1,
n=0, X.sup.2=allyl, p=1, and o=0, such that the compound (44)
represents a dimeric structure containing two DODOyl fragments
corresponding to the active moieties of ajoene, and
X.sup.3=--(CH.sub.2).sub.q--, where q is selected from the group
consisting of 1, 2, 3, 4, 5, and 6.
[0289] The foregoing detailed description and representative
examples have been provided solely by way of explanation and
illustration, and are not intended to limit the scope of the
appended claims. Many variations in the presently preferred
embodiments illustrated herein will be obvious to one of ordinary
skill in the art, and remain within the scope of the appended
claims and their equivalents.
* * * * *